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Targeting Trim69 alleviates high fat diet (HFD)-induced hippocampal injury in mice by inhibiting apoptosis and inflammation through ASK1 inactivation
Biochemical and Biophysical Research Communications 515 (2019) 658e664
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Targeting Trim69 alleviates high fat diet (HFD)-induced hippocampal injury in mice by inhibiting apoptosis and inflammation through ASK1 inactivation Lin-Juan Li a, b, 1, Jun-Chen Zheng c, 1, Rui Kang d, Jian-Qun Yan a, * a
Department of Physiology and Pathophysiology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi, 710061, China Department of General Internal Medicine, Yan'an University Affiliated Hospital, Yan'an, Shaanxi, 716000, China Department of Cardiovascular Medicine Center, Yan'an University Affiliated Hospital, Yan'an, Shaanxi, 716000, China d Department of Respiratory Medicine, Yan'an University Affiliated Hospital, Yan'an, Shaanxi, 716000, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2019 Accepted 3 May 2019 Available online 6 June 2019
The prevalence of obesity is growing, and high fat diet (HFD)-induced obesity can alter the brain and cognition. However, the link between HFD, hippocampal function, and inflammation is still not fully understood. Tripartite motif (TRIM) family has been implicated in various cellular processes, such as apoptosis, neurogenesis, and innate immune responses. Trim69, a member of TRIM family, was investigated in the present study to determine its role in HFD-induced hippocampal damage. Here, we first found that hippocampal Trim69 expression was markedly down-regulated in wild-type (WT) mice challenged with HFD. Trim69 knockout (KO) mice exhibited an exaggerated version of the metabolic disorder after HFD challenge, as evidenced by their increased body weight and elevated insulin resistance. HFD-induced hippocampal injury was further aggravated by Trim69 deletion, as confirmed by the reduced survival of neurons and increased level of apoptotic cell death. In addition, the inflammatory response triggered by HFD was more pronounced in the hippocampi of Trim69-KO mice after blockage of the activation of the nuclear factor kappa B (NF-kB) signaling pathway. Phosphorylation of mitogenactivated protein kinase (MAPK) kinase 4 (MKK4), MKK7, and c-Jun N-terminal kinase (JNK) in the hippocampi of HFD-challenged mice was intensified by the loss of Trim69. Hippocampal-apoptosissignal-regulating kinase 1 (ASK1) phosphorylation was also found to be up-regulated by HFD, especially in mice with Trim69 deletion. Of note, we found that Trim69 directly interacted with and deubiquitinated ASK1 in microglial cells. Microglial cell-specific suppression of Trim69 exacerbated inflammation and apoptosis in response to lipopolysaccharide (LPS). Trim69 over-expression markedly alleviated LPS-induced inflammatory response and apoptotic cell death in microglial cells. Together, these results indicated that Trim69 might be a functionally essential inhibitor of ASK1 activation during the pathogenesis of hippocampal inflammation and apoptosis, and it could serve as a novel molecular target for obesity-associated brain damage. © 2019 Published by Elsevier Inc.
Keywords: Obesity Trim69 Hippocampus Inflammation and apoptosis ASK1
1. Introduction Obesity has become a worldwide epidemic, and the correlations between human obesity and both cognitive decline and central nervous system injury have been established [1]. Moreover,
* Corresponding author. Xi'an Jiaotong University School of Medicine, 76 Yanta West Road, Yanta District, Xi'an, Shaanxi Province, 710061, China. E-mail addresses: [email protected], [email protected] (J.-Q. Yan). 1 Lin-Juan Li and Jun-Chen Zheng are the co-first authors. https://doi.org/10.1016/j.bbrc.2019.05.027 0006-291X/© 2019 Published by Elsevier Inc.
metabolic alterations induced by obesity have been found to be related to abnormal cognition and neuronal changes [2]. A large number of clinical and rodent studies have linked hippocampal injury to cognitive impairments in individuals with obesity [3]. Inflammation and apoptosis play crucial roles in neuronal injury and death in brains subjected to various types of assault, such as hyperoxia, ischemia, and HFD-induced metabolic disorder brain injury [4,5]. Recently, dysregulated production and secretion of cytokines, including tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and IL-1b, have been implicated in the progression of hippocampal damage, contributing to cognitive impairment ultimately
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[6]. Apoptotic cell death is also involved in the modulation of synaptic activities, cognitive processes, and learning and memory [7]. However, the molecular mechanism underlying diet-induced hippocampal injury is not completely understood, hindering the development of effective therapeutic strategies for this disease [8]. Tripartite motif 69 (Trim69), a member of the TRIM family of proteins, has been reported to participate in diverse biological processes, including cell growth, death, differentiation, immune responses, and tumor progression [9]. TRIM family proteins contain a RING finger domain, which may contribute to E3 ubiquitin ligase activity [10]. Trim69 is evolutionarily conserved in vertebrates. Trim69 knockdown stimulates p53 signaling and promotes apoptosis during embryogenesis [11]. In addition, Trim69 expression is involved in zebrafish brain development partly through regulating JNK activation [12]. The activity of JNK plays a crucial role in mediating the progression of inflammatory response and apoptosis [13]. Trim69 might be involved in metabolic stressinduced hippocampal injury associated with JNK signaling. However, the precisely functional and biological relevance of Trim69 in hippocampal injury and associated pathologies remains to be established. In the study, we found Trim69 expression to be up-regulated in hippocampus samples of HFD-fed mice. Trim69 knockout markedly accelerated metabolic disorder in HFD-challenged mice and hippocampal damage. Furthermore, HFD-induced inflammation and apoptosis in the hippocampi of mice were promoted by the loss of Trim69 through elevation of the NF-kB and JNK signaling pathways. Notably, we established the therapeutic potential of Trim69regulated ASK1 deubiquitination in microglia cell inflammation, which was implicated in the progression of HFD-induced hippocampal injury. These data show a new molecular mechanism revealing the modulation of ASK1 by Trim69 during hippocampal damage, indicating a promising therapeutic target for the treatment of this disease.
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kept at 37 C in a humidified incubator with 5% CO2. Lipopolysaccharide (LPS) was obtained from Sigma Aldrich (USA) for in vitro stimulation. To knock down Trim69 expression, three mouse Trim69-specific short hairpin RNA (shRNA)-expressing (shTrim69) constructs were purchased from OriGene (Rockville, MD, USA). LPS was purchased from Sigma Aldrich to stimulate the cells. 2.3. Real time-quantitative PCR (RT-qPCR) and western blotting Total mRNA was extracted from hippocampus tissues and BV2 cells and reverse-transcribed into cDNA according to the manufacturer's instructions (Takara, Dalian, China). RT-qPCR was conducted with SYBR Green PCR Master Mix (Roche) as previously indicated [14]. Relative expression was normalized to GAPDH expression. The primers used in the study were exhibited in Supplementary Table 1. As for Western blot analysis, the hippocampus samples or cultured cells were lysed in the RIPA buffer, and 1% Triton X-100 on ice. After centrifugation at 12, 000 g and 4 C for 20 min, the supernatant was subjected to 10% SDS-PAGE and electro-transferred onto a PVDF membrane (Millipore, USA). Then, transferred proteins were incubated with primary antibodies (Supplementary Table 2) and secondary antibodies (Pierce, USA). Signals were detected with ECL (Pierce). 2.4. Adenovirus infection Adenovirus preparation and infection in cells were performed as previously indicated [15]. Briefly, adenovirus vectors expressing Trim69 (Ad-Trim69) or GFP (Ad-GFP, used as control) were used. Recombinant adenoviruses were produced by the AdEasy vector kit (Stratagene, USA). Adenoviruses with a multiplicity of infection (MOI) of 100 were applied to infect BV2 cells. The infected cells were then subjected to LPS stimulation for 24 h after infection. 2.5. Biochemical analysis
2. Materials and methods 2.1. Animals and models The Animal Care and Use Committee of Xi'an Jiaotong University School of Medicine (Shaanxi, China) approved all animal experiments. Human care was provided following the criteria indicated in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. Male, 8e10 weeks old, wide type mice (WT) were purchased from SLAC Laboratory animal co. LTD. (Shanghai, China). Trim69 knockout (KO) mice were purchased from Shanghai Biomodel Organism Science & Technology Development Co.,Ltd. (Shanghai). All mice were nurtured in a temperature-controlled environment under a 12 h light/dark time cycle. HFD (D12492, Research Diets, NJ, USA) and normal chow diet (NCD; D12450B, Research Diets) were subjected to mice continuously for 16 weeks and control mice, respectively. 60 animals were divided into four groups (n ¼ 15/group): WT/NCD; KO/NCD; WT/HFD; and KO/HFD. All mice had free access to food and water. Body weights were measured every week. After HFD challenge for 16 weeks, blood was obtained from eyepit, and the mice were sacrificed. Hippocampus was collected on ice and stored at 80 C for further studies. 2.2. Cells and culture Mouse microglia cells, BV2, were purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, penicillin (100 U/ml)/streptomycin (100 mg/ml) (Invitrogen, USA). Cultures were
Fasting blood insulin levels were measured using ELISA (Millipore, USA). The levels of TNF-a, IL-1b and IL-6 in serum were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems, USA) following the supplier's instructions. 2.6. Oral glucose tolerance test (OGTT) Blood glucose levels in the tail vein were measured at 0, 15, 30, 60 and 120 min after the glucose (2 g/kg body weight) injection using the Ascensia ELITE blood glucose meter (Bayer Diagnostics Europe Ltd.; Dublin, Ireland). 2.7. Immunohistochemical analysis Brain samples were removed and post-fixed in 4% paraformaldehyde overnight at 4 C, then transferred to 30% sucrose solution in PBS, and frozen with dry ice before storage at 80 C until sectioning. A 1-in-5 series of sections containing the hippocampus was cut at 40 mm. Tissue sections were then subjected to Nissl staining with cresyl violet [16]. To detect apoptotic cell death, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed as a positive indicator used in determination of apoptosis. One-step TUNEL kit (Beyotime Institute, Nanjing, China) was used as indicated by the manufacturer. Immunohistochemistry was performed through the avidin-biotinperoxidase complex method [17]. The primary antibody used in the study included Caspase-3 (1:150; Abcam). Immunopositive cells in which the reaction product were quantified under a light microscope.
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2.8. Immunofluorescent analysis Frozen brain sections were subjected to immunofluorescence staining as previously shown [18]. Primary antibodies included Trim69 (Sigma Aldrich), NeuN (Abcam, USA), Iba-1 (Abcam) and pNF-kB (Abcam). Immunofluorescence was performed with Alexa Fluor 488- or 594-labeled secondary antibodies (Abcam) and DAPI (Sigma Aldrich) to identify cell nuclei. Representative images were captured using Confocal Microscopes (Nikon, Japan). 2.9. Plasmid constructs and immunoprecipitation (IP) assay All plasmids were constructed by Research Science Bio
(Shanghai, China). Plasmids encoding full-length mouse FlagTrim69 were obtained through cloning the cDNA of Trim69 into the psi-Flag vectors. Plasmids encoding full-length mouse HA-ASK1 were obtained by cloning the indicated cDNA of ASK1 into pcDNA5HA vector. Ubiquitin was cloned into the Myc-tagged pcDNA5 vectors that the ubiquitin encoded by the plasmid with an N-terminal Myc tag. IP assays of Trim69 and ASK1 were performed to determine the interaction condition as previously described in cultured BV2 cells cotransfected with the indicated reconstructs [19]. Then, the cells were lysed and centrifuged. Subsequently, each sample was incubated with Protein A/G-agarose beads (Pierce) and primary antibodies (Supplementary Table 2). The immunocomplexes was washed by IP buffer for immunoblotting analysis with
Fig. 1. Effects of Trim69 on metabolic disorder and hippocampus injury in mice challenged with HFD. (A) RT-qPCR and (B,C) Western blot analysis of Trim69 expression in hippocampus of HFD-fed mice. (D) Immunofluorescent analysis of Trim69 and NeuN in hippocampus of HFD-challenged mice. (E) Body weight of mice after HFD treatment. Calculation of (F) fasting blood glucose levels and (G) insulin levels. (H) OGTT results were showed. (I) Nissl staining of CA3 and CA1 in hippocampus of mice. (J) TUNEL staining (up panel) and immunohistochemical analysis of Caspase-3 (down panel) in hippocampus of mice. Plots were mean ± SEM values. *p < 0.05 and **p < 0.01; þp < 0.05 vs HFD/WT group.
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the exhibited primary antibodies and corresponding secondary antibodies.
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two groups; one-way analysis of variance (ANOVA) was used for multiple comparisons with Bonferroni post hoc analysis. P < 0.05 was considered to be significant.
2.10. Ubiquitination assay BV2 cells were pre-treated with LPS (100 ng/ml) for 24 h and MG132 for 6 h. Endogenous ASK1 was immunoprecipitated from protein lysate using the ASK1 antibody and then immunoblotted with the anti-ubiquitin antibody to capture polyubiquitinated ASK1. BV2 cells were co-transfected with Myc-Ub, HA-ASK1 or Flag-Trim69 plasmids. 24 h after transfection, the cells were treated with LPS for further 24 h. Then, MG132 was added to the culture medium for 6 h prior to harvesting. Cells were split into two aliquots, one for immunoblotting and the other for an ubiquitination assay. For ubiquitination assay, the anti-HA antibody was used to immunoprecipitate ASK1 and then detected the ubiquitination using a Myc-tag antibody. 2.11. Flow cytometry analysis Annexin V-FITC apoptosis detection kit (Sigma Aldrich) was used following the manufactured protocols on a flow cytometer (Becton and Dickinson, USA) to analyze the populations of cells. 2.12. Statistical analysis All values were expressed as the mean ± SEM using GraphPad Prism 6.0 software (GraphPad Software, San Diego, USA). Statistical analysis was performed using the two-tailed Student's t-test for
3. Results 3.1. Effects of Trim69 on metabolic disorders and hippocampal injury in mice challenged with HFD To investigate the effects of Trim69 on metabolic-stress-induced hippocampal injury, the expression of Trim69 was measured. As shown in Fig. 1AeC, hippocampal Trim69 expression from mRNA and protein levels was markedly down-regulated in HFD-fed mice. Immunofluorescent analysis suggested that both Trim69 and NeuN expression levels were reduced by HFD challenge (Fig. 1D). Then, HFD-treated mice with or without Trim69 knockout were used to further explore the role of Trim69 in regulating hippocampal damage. As shown in Fig. 1E, HFD feeding led to a significant increase in the body weight of WT mice, which was more pronounced in Trim69-KO mice. This showed that HFD-induced elevation of fasting blood glucose and insulin levels was enhanced by the deficiency of Trim69 (Fig. 1F and G). OGTT analysis further indicated that Trim69 ablation markedly exacerbated HFD-induced insulin resistance in HFD-fed mice (Fig. 1H). Nissl staining showed that HFD treatment led to significant reduction in the number of survival neurons in CA3 and CA1 areas, which was further decreased by Trim69 deficiency (Fig. 1I). We also found the number of TUNELand caspase-3-positive cells in the hippocampi of mice to be aggravated in HFD-challenged mice with Trim69 deletion (Fig. 1J).
Fig. 2. Trim69 deficiency elevates inflammatory response in hippocampus of HFD-treated mice. (A) ELISA and (B) RT-qPCR analysis of TNF-a, IL-1b and IL-6 in serum and hippocampus tissues, respectively. (C) Western blot analysis of p-IkBa and p-NF-kB in hippocampus of mice. (D) Immunofluorescent analysis of p-NF-kB and Iba-1 in hippocampus of HFD-fed mice with or without Trim69 expression. (E) Western blot analysis of p-MKK4, p-MKK7 and p-JNK in hippocampus of mice. Plots were mean ± SEM values. *p < 0.05 and ** p < 0.01.
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These findings indicated that Trim69 played an essential role in metabolic stress-induced metabolic disorder and hippocampal damage.
3.2. Trim69 deficiency elevates inflammatory response in hippocampus of HFD-treated mice ELISA and RT-qPCR analysis indicated that HFD-enhanced systematic and hippocampal TNF-a, IL-1b, and IL-6 expression were further promoted in HFD-fed mice with Trim69 knockout (Fig. 2A and B). Western blot results indicated that the expression of phosphorylated IkBa and NF-kB in the hippocampus of HFD mice was markedly elevated by Trim69 deficiency (Fig. 2C). This indicated Trim69 expression plays a role in HFD-induced inflammatory response in the hippocampus of mice. Iba-1 is a hallmark of microglia activation, playing an essential role in regulating neuroinflammation, and this has been investigated [20,21]. Immunofluorescent analysis suggested that Trim69 ablation visibly upregulated the expression of p-NF-kB and Iba-1 in the hippocampus of HFD-fed mice over the HFD group (Fig. 2D). Given that Trim69 expression was correlated with JNK activation, we then investigated JNK signaling. As shown in Fig. 2E, the levels of expression of p-MKK4, p-MKK7, and p-JNK in hippocampi of mice were highly induced by HFD feeding, while being further promoted in Trim69-KO mice. These results suggested that Trim69-regulated hippocampal damage in HFD-fed mice was closely associated with the inflammatory response.
3.3. Trim69 directly interacts with ASK1 ASK1 was found to drive the inflammatory response in tissues mainly through a down-streaming JNK pathway. ASK1 activation was measured to further calculate the effects of Trim69 on HFDinduced hippocampal inflammation. As expected, Trim69 knockout markedly accelerated the expression of p-ASK1 in the hippocampus of HFD-fed mice (Fig. 3A). In this way, Trim69 showed a suppressive role in the ASK1 activation. We used microglial cells, which play a critical role in regulation of inflammation in the central nervous system, in subsequent studies [22]. We found that Trim69 inhibited ASK1 phosphorylation in a dose-dependent manner in LPS-incubated microglial cells transfected with varying amounts of Flag-Trim69 plasmids (Fig. 3B). Furthermore, specific interaction between Trim69 and ASK1 was observed by co-IP assays (Fig. 3C). ASK1 activation is associated with its polyubiquitination [23]. Within expectation, LPS stimulation significantly induced ASK1 ubiquitination and up-regulated ASK1 phosphorylation in BV2 cells (Fig. 3D). Over-expression of Trim69 in microglial cells led to substantial deubiquitination of ASK1 and suppression of its activation in response to LPS (Fig. 3E). 3.4. Trim69 expression protects against inflammation and apoptosis in microglia cells stimulated by LPS The expression of Trim69 was knocked down after infection with its targeting shRNAs (Fig. 4A). Also, the phosphorylation of ASK1 was inhibited by the suppression of Trim69 in microglial cells
Fig. 3. Trim69 directly interacts with ASK1. (A) Western blot analysis of p-ASK1 in hippocampus of mice from the indicated groups. (B) Western blot analysis of ASK1 and p-ASK1 in BV2 cells transfected with different amounts of Flag-tagged Trim69. (C) CO-IP results of BV2 cells transfected with Flag-tagged Trim69 or HA-ASK1. Flag and HA antibodies were applied for western blotting probes. (D) Western blot analysis of ASK1 ubiquitination and total ASK1 after IP for ASK1 (up panel), and p-ASK1 and ASK1 in lysate of BV2 cells stimulated with or without LPS (100 ng/ml) for 24 h. (E) Western blot analysis of ASK1 ubiquitination in BV2 cells with the transfection of plasmids as shown in the absence or presence of LPS (100 ng/ml) for 24 h. Plots were mean ± SEM values. *p < 0.05 and **p < 0.01.
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Fig. 4. Trim69 expression protects against inflammation and apoptosis in microglia cells stimulated by LPS. (A) Western blot analysis of Trim69 in BV2 cells transfected with different Trim69-targeting shRNAs (shTrim69) showing different inhibition efficacy. (B) Western blot analysis of p-ASK1 in BV2 cells transfected with different Trim69-targeting shRNAs with or without stimulation of LPS (100 ng/ml, 24 h). (C) Western blot analysis of p-ASK1, p-MKK4, p-MKK7, p-JNK and p-NF-kB in BV2 cells transfected with Trim69targeting shRNA with or without stimulation of LPS (100 ng/ml, 24 h). (D) RT-qPCR analysis of TNF-a, IL-1b and IL-6 and (E) flow cytometry analysis for apoptotic cell death in shTrim69-transfected BV2 cells with or without LPS stimulation (100 ng/ml) for 24 h. (F) Western blot analysis of p-ASK1, p-MKK4, p-MKK7, p-JNK and p-NF-kB in BV2 cells infected with Trim69-overexpressing Ad-Trim69 with or without stimulation of LPS (100 ng/ml, 24 h). (G) RT-qPCR analysis of TNF-a, IL-1b and IL-6 and (H) flow cytometry analysis for apoptotic cell death in BV2 cells infected with Trim69-overexpressing Ad-Trim69 in the absence or presence of LPS (100 ng/ml, 24 h). (I) Schematic of the role and regulatory mechanisms of Trim69 on HFD-induced hippocampus injury. Plots were mean ± SEM values. *p < 0.05 and **p < 0.01.
(Fig. 4B). Then, the shTrim69-3 was further applied to microglial cells for Trim69 inhibition. Western blot analysis suggested that the suppression of Trim69 further promoted the expression of p-ASK1, p-MKK4, p-MKK7, p-JNK, and p-NF-kB in BV2 cells upon LPS stimulation (Fig. 4C). Consistently, in LPS-incubated microglial cells, shTrim69 infection markedly increased the mRNA levels of TNF-a, IL-1b, and IL-6, as well as the rate of apoptotic cell death (Fig. 4D and E). In contrast, over-expressing Trim69 decreased the expression of p-ASK1, p-MKK4, p-MKK7, p-JNK, and p-NF-kB in microglial cells with LPS stimulation, along with subsequent down-regulation of TNF-a, IL-1b, and IL-6 by Western blot and RT-qPCR analysis (Fig. 4F and G). LPS-induced apoptosis in BV2 cells stimulated by LPS was visibly alleviated by Trim69 over-expression (Fig. 4H). These results indicated that Trim69 protected cells from inflammation and apoptosis in microglial cells stimulated with LPS. 4. Discussion In this study, we established the essential role of Trim69 in regulating the progression of hippocampal injury induced by HFD. We found Trim69 expression to be markedly up-regulated in the hippocampi of HFD-fed mice. Trim69 deficiency significantly accelerated HFD-induced metabolic disorder and hippocampal injury, which was closely associated with the progression of inflammation and apoptosis by activating NF-kB and JNK signaling
pathways. Trim69 expression negatively regulated ASK1 phosphorylation in the hippocampi of HFD-fed mice, and in LPSstimulated microglial cells. We verified that Trim69 directly interacted with and deubiquitinated ASK1 in microglial cells. The protective role of Trim69 against inflammation and apoptosis was further confirmed in LPS-incubated microglial cells. Our findings demonstrated that treatments promoting the deubiquitinating activity of Trim69 to inhibit ASK1 activation were promising leads as hippocampal injury therapies (Fig. 4I). The tripartite motif family proteins (TRIMs) share three conserved domains, an N-terminal Really Interesting New Gene (RING) domain, one or two B-Boxes (B1/B2), and a coiled-coil (CC) domain. These play crucial roles in various cellular processes, including apoptosis, neurogenesis, and innate immune responses [9,10]. Trim69, as a TRIM family member, is an IFN-inducible virus restriction factor that was found to be involved in apoptosis, tumor control, and zebrafish development [12,24,25]. Recently, TRIM69 was found to significantly suppress ultraviolet B (UVB)-induced apoptosis and oxidative stress. It could also interact with p53 and induce its ubiquitination, and functioned as an inhibitory factor during cataractogenesis [11]. Trim69 also regulates zebrafish brain development, which was largely associated with the activation of JNK signaling [12]. Inflammation and apoptosis are two critical pathologies that contribute to the progression of hippocampal injury associated with obesity [4,5,26,27]. In line with previous
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studies, we here found that HFD led to significant increases in the expression of pro-inflammatory cytokines, including TNF-a, IL-1b, and IL-6. This process took place largely by increasing the activation of IkBa/NF-kB signaling pathway. This is the first report to show that HFD-stimulated NF-kB activation was further promoted by the Trim69 deficiency. The JNK signaling pathway also plays a significant role in promoting the NF-kB signaling to promote the development of inflammatory response under diverse pathological processes [13,28]. JNK could be activated by MAPK kinases (MAPKKs), including mitogen-activated protein kinases 4 and 7 (MKK4 and MKK7) [29]. MAPKKs can in turn be activated by apoptosis signal regulating kinase group (ASK1), also known as MAP3K5 [30]. Of the MAPKKK molecules, ASK1 was found to be ubiquitously expressed and activates the MKK4/7-JNK signaling cascades [31]. ASK1 is a crucial facilitator and therapeutic target for preventing brain injury associated with obesity [32]. In our study, we found that HFD treatment markedly induced the phosphorylation of MKK4, MKK7, and JNK in the hippocampi of mice, and this can be further accelerated by Trim69 deletion, along with enhanced ASK1 phosphorylation (Fig. 4I). These results were consistent with the alterations of inflammatory response. IP analysis further demonstrated that Trim69 could directly interact with ASK1 and negatively regulate ASK1 activation. ASK1 polyubiquitination is related to its activation [33,34]. Our in vitro experiments suggested that LPS treatment of microglial cells led to significant ASK1 ubiquitination as well as upregulated levels of phosphorylated ASK1. Trim69 over-expression in microglial cells caused substantial ASK1 deubiquitination and suppression of its activation upon LPS stimulation. These findings further indicated that ASK1 modulation by Trim69 took place through activation-associated deubiquitination during the development of hippocampal injury induced by HFD. In conclusion, our findings here provide the first solid evidence that the polyubiquitination-dependent activation of ASK1 signaling pathway in response to LPS can be inhibited by Trim69. Furthermore, the Trim69/ASK1 pathway showed protective effects to prevent inflammation and apoptotic cell death, which might be involved in the treatment of hippocampal injury induced by obesity or associated metabolic stresses (Fig. 4I). However, further studies are still warranted to explore the effects of Trim69 on more specific mediators of hippocampal injury for developing effective treatments. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.027. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.027 References [1] M. Kivipelto, et al., Obesity and vascular risk factors at midlife and the risk of dementia and Alzheimer disease, Arch. Neurol. 62 (10) (2005) 1556e1560. [2] B.E. Levin, The obesity epidemic: metabolic imprinting on genetically susceptible neural circuits, Obes. Res. 8 (4) (2000) 342e347. [3] J.P. Thaler, et al., Obesity is associated with hypothalamic injury in rodents and humans, J. Clin. Investig. 122 (1) (2012) 153e162. [4] S.D. Bilbo, et al., Enduring consequences of maternal obesity for brain
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[27] M.D. Niculescu, et al., High fat diet-induced maternal obesity alters fetal hippocampal development, Int. J. Dev. Neurosci. 27 (7) (2009) 627e633. [28] Y.W. Ki, et al., JNK and p38 MAPK regulate oxidative stress and the inflammatory response in chlorpyrifos-induced apoptosis, Toxicol. Lett. 218 (3) (2013) 235e245. [29] W. Haeusgen, et al., The bottleneck of JNK signaling: molecular and functional characteristics of MKK4 and MKK7, Eur. J. Cell Biol. 90 (6e7) (2011) 536e544. [30] T. Liang, et al., Curcumin induced human gastric cancer BGC-823 cells apoptosis by ROS-mediated ASK1-MKK4-JNK stress signaling pathway, Int. J. Mol. Sci. 15 (9) (2014) 15754e15765. [31] K. Takeda, et al., Roles of MAPKKK ASK1 in stress-induced cell death, Cell Struct. Funct. 28 (1) (2003) 23e29. [32] K. Toyama, et al., ASK1 is involved in cognitive impairment caused by longterm high-fat diet feeding in mice, Sci. Rep. 5 (2015) 10844. [33] Y. 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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Targeting Trim69 alleviates high fat diet (HFD)-induced hippocampal injury in mice by inhibiting apoptosis and inflammation through ASK1 inactivation Lin-Juan Li a, b, 1, Jun-Chen Zheng c, 1, Rui Kang d, Jian-Qun Yan a, * a
Department of Physiology and Pathophysiology, Xi'an Jiaotong University School of Medicine, Xi'an, Shaanxi, 710061, China Department of General Internal Medicine, Yan'an University Affiliated Hospital, Yan'an, Shaanxi, 716000, China Department of Cardiovascular Medicine Center, Yan'an University Affiliated Hospital, Yan'an, Shaanxi, 716000, China d Department of Respiratory Medicine, Yan'an University Affiliated Hospital, Yan'an, Shaanxi, 716000, China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2019 Accepted 3 May 2019 Available online 6 June 2019
The prevalence of obesity is growing, and high fat diet (HFD)-induced obesity can alter the brain and cognition. However, the link between HFD, hippocampal function, and inflammation is still not fully understood. Tripartite motif (TRIM) family has been implicated in various cellular processes, such as apoptosis, neurogenesis, and innate immune responses. Trim69, a member of TRIM family, was investigated in the present study to determine its role in HFD-induced hippocampal damage. Here, we first found that hippocampal Trim69 expression was markedly down-regulated in wild-type (WT) mice challenged with HFD. Trim69 knockout (KO) mice exhibited an exaggerated version of the metabolic disorder after HFD challenge, as evidenced by their increased body weight and elevated insulin resistance. HFD-induced hippocampal injury was further aggravated by Trim69 deletion, as confirmed by the reduced survival of neurons and increased level of apoptotic cell death. In addition, the inflammatory response triggered by HFD was more pronounced in the hippocampi of Trim69-KO mice after blockage of the activation of the nuclear factor kappa B (NF-kB) signaling pathway. Phosphorylation of mitogenactivated protein kinase (MAPK) kinase 4 (MKK4), MKK7, and c-Jun N-terminal kinase (JNK) in the hippocampi of HFD-challenged mice was intensified by the loss of Trim69. Hippocampal-apoptosissignal-regulating kinase 1 (ASK1) phosphorylation was also found to be up-regulated by HFD, especially in mice with Trim69 deletion. Of note, we found that Trim69 directly interacted with and deubiquitinated ASK1 in microglial cells. Microglial cell-specific suppression of Trim69 exacerbated inflammation and apoptosis in response to lipopolysaccharide (LPS). Trim69 over-expression markedly alleviated LPS-induced inflammatory response and apoptotic cell death in microglial cells. Together, these results indicated that Trim69 might be a functionally essential inhibitor of ASK1 activation during the pathogenesis of hippocampal inflammation and apoptosis, and it could serve as a novel molecular target for obesity-associated brain damage. © 2019 Published by Elsevier Inc.
Keywords: Obesity Trim69 Hippocampus Inflammation and apoptosis ASK1
1. Introduction Obesity has become a worldwide epidemic, and the correlations between human obesity and both cognitive decline and central nervous system injury have been established [1]. Moreover,
* Corresponding author. Xi'an Jiaotong University School of Medicine, 76 Yanta West Road, Yanta District, Xi'an, Shaanxi Province, 710061, China. E-mail addresses: [email protected], [email protected] (J.-Q. Yan). 1 Lin-Juan Li and Jun-Chen Zheng are the co-first authors. https://doi.org/10.1016/j.bbrc.2019.05.027 0006-291X/© 2019 Published by Elsevier Inc.
metabolic alterations induced by obesity have been found to be related to abnormal cognition and neuronal changes [2]. A large number of clinical and rodent studies have linked hippocampal injury to cognitive impairments in individuals with obesity [3]. Inflammation and apoptosis play crucial roles in neuronal injury and death in brains subjected to various types of assault, such as hyperoxia, ischemia, and HFD-induced metabolic disorder brain injury [4,5]. Recently, dysregulated production and secretion of cytokines, including tumor necrosis factor-a (TNF-a), interleukin-6 (IL-6), and IL-1b, have been implicated in the progression of hippocampal damage, contributing to cognitive impairment ultimately
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[6]. Apoptotic cell death is also involved in the modulation of synaptic activities, cognitive processes, and learning and memory [7]. However, the molecular mechanism underlying diet-induced hippocampal injury is not completely understood, hindering the development of effective therapeutic strategies for this disease [8]. Tripartite motif 69 (Trim69), a member of the TRIM family of proteins, has been reported to participate in diverse biological processes, including cell growth, death, differentiation, immune responses, and tumor progression [9]. TRIM family proteins contain a RING finger domain, which may contribute to E3 ubiquitin ligase activity [10]. Trim69 is evolutionarily conserved in vertebrates. Trim69 knockdown stimulates p53 signaling and promotes apoptosis during embryogenesis [11]. In addition, Trim69 expression is involved in zebrafish brain development partly through regulating JNK activation [12]. The activity of JNK plays a crucial role in mediating the progression of inflammatory response and apoptosis [13]. Trim69 might be involved in metabolic stressinduced hippocampal injury associated with JNK signaling. However, the precisely functional and biological relevance of Trim69 in hippocampal injury and associated pathologies remains to be established. In the study, we found Trim69 expression to be up-regulated in hippocampus samples of HFD-fed mice. Trim69 knockout markedly accelerated metabolic disorder in HFD-challenged mice and hippocampal damage. Furthermore, HFD-induced inflammation and apoptosis in the hippocampi of mice were promoted by the loss of Trim69 through elevation of the NF-kB and JNK signaling pathways. Notably, we established the therapeutic potential of Trim69regulated ASK1 deubiquitination in microglia cell inflammation, which was implicated in the progression of HFD-induced hippocampal injury. These data show a new molecular mechanism revealing the modulation of ASK1 by Trim69 during hippocampal damage, indicating a promising therapeutic target for the treatment of this disease.
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kept at 37 C in a humidified incubator with 5% CO2. Lipopolysaccharide (LPS) was obtained from Sigma Aldrich (USA) for in vitro stimulation. To knock down Trim69 expression, three mouse Trim69-specific short hairpin RNA (shRNA)-expressing (shTrim69) constructs were purchased from OriGene (Rockville, MD, USA). LPS was purchased from Sigma Aldrich to stimulate the cells. 2.3. Real time-quantitative PCR (RT-qPCR) and western blotting Total mRNA was extracted from hippocampus tissues and BV2 cells and reverse-transcribed into cDNA according to the manufacturer's instructions (Takara, Dalian, China). RT-qPCR was conducted with SYBR Green PCR Master Mix (Roche) as previously indicated [14]. Relative expression was normalized to GAPDH expression. The primers used in the study were exhibited in Supplementary Table 1. As for Western blot analysis, the hippocampus samples or cultured cells were lysed in the RIPA buffer, and 1% Triton X-100 on ice. After centrifugation at 12, 000 g and 4 C for 20 min, the supernatant was subjected to 10% SDS-PAGE and electro-transferred onto a PVDF membrane (Millipore, USA). Then, transferred proteins were incubated with primary antibodies (Supplementary Table 2) and secondary antibodies (Pierce, USA). Signals were detected with ECL (Pierce). 2.4. Adenovirus infection Adenovirus preparation and infection in cells were performed as previously indicated [15]. Briefly, adenovirus vectors expressing Trim69 (Ad-Trim69) or GFP (Ad-GFP, used as control) were used. Recombinant adenoviruses were produced by the AdEasy vector kit (Stratagene, USA). Adenoviruses with a multiplicity of infection (MOI) of 100 were applied to infect BV2 cells. The infected cells were then subjected to LPS stimulation for 24 h after infection. 2.5. Biochemical analysis
2. Materials and methods 2.1. Animals and models The Animal Care and Use Committee of Xi'an Jiaotong University School of Medicine (Shaanxi, China) approved all animal experiments. Human care was provided following the criteria indicated in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health. Male, 8e10 weeks old, wide type mice (WT) were purchased from SLAC Laboratory animal co. LTD. (Shanghai, China). Trim69 knockout (KO) mice were purchased from Shanghai Biomodel Organism Science & Technology Development Co.,Ltd. (Shanghai). All mice were nurtured in a temperature-controlled environment under a 12 h light/dark time cycle. HFD (D12492, Research Diets, NJ, USA) and normal chow diet (NCD; D12450B, Research Diets) were subjected to mice continuously for 16 weeks and control mice, respectively. 60 animals were divided into four groups (n ¼ 15/group): WT/NCD; KO/NCD; WT/HFD; and KO/HFD. All mice had free access to food and water. Body weights were measured every week. After HFD challenge for 16 weeks, blood was obtained from eyepit, and the mice were sacrificed. Hippocampus was collected on ice and stored at 80 C for further studies. 2.2. Cells and culture Mouse microglia cells, BV2, were purchased from ATCC (Manassas, VA, USA). Cells were cultured in DMEM (Gibco, USA) with 10% fetal bovine serum (FBS, Gibco), 2 mM L-glutamine, penicillin (100 U/ml)/streptomycin (100 mg/ml) (Invitrogen, USA). Cultures were
Fasting blood insulin levels were measured using ELISA (Millipore, USA). The levels of TNF-a, IL-1b and IL-6 in serum were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) (R&D Systems, USA) following the supplier's instructions. 2.6. Oral glucose tolerance test (OGTT) Blood glucose levels in the tail vein were measured at 0, 15, 30, 60 and 120 min after the glucose (2 g/kg body weight) injection using the Ascensia ELITE blood glucose meter (Bayer Diagnostics Europe Ltd.; Dublin, Ireland). 2.7. Immunohistochemical analysis Brain samples were removed and post-fixed in 4% paraformaldehyde overnight at 4 C, then transferred to 30% sucrose solution in PBS, and frozen with dry ice before storage at 80 C until sectioning. A 1-in-5 series of sections containing the hippocampus was cut at 40 mm. Tissue sections were then subjected to Nissl staining with cresyl violet [16]. To detect apoptotic cell death, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed as a positive indicator used in determination of apoptosis. One-step TUNEL kit (Beyotime Institute, Nanjing, China) was used as indicated by the manufacturer. Immunohistochemistry was performed through the avidin-biotinperoxidase complex method [17]. The primary antibody used in the study included Caspase-3 (1:150; Abcam). Immunopositive cells in which the reaction product were quantified under a light microscope.
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2.8. Immunofluorescent analysis Frozen brain sections were subjected to immunofluorescence staining as previously shown [18]. Primary antibodies included Trim69 (Sigma Aldrich), NeuN (Abcam, USA), Iba-1 (Abcam) and pNF-kB (Abcam). Immunofluorescence was performed with Alexa Fluor 488- or 594-labeled secondary antibodies (Abcam) and DAPI (Sigma Aldrich) to identify cell nuclei. Representative images were captured using Confocal Microscopes (Nikon, Japan). 2.9. Plasmid constructs and immunoprecipitation (IP) assay All plasmids were constructed by Research Science Bio
(Shanghai, China). Plasmids encoding full-length mouse FlagTrim69 were obtained through cloning the cDNA of Trim69 into the psi-Flag vectors. Plasmids encoding full-length mouse HA-ASK1 were obtained by cloning the indicated cDNA of ASK1 into pcDNA5HA vector. Ubiquitin was cloned into the Myc-tagged pcDNA5 vectors that the ubiquitin encoded by the plasmid with an N-terminal Myc tag. IP assays of Trim69 and ASK1 were performed to determine the interaction condition as previously described in cultured BV2 cells cotransfected with the indicated reconstructs [19]. Then, the cells were lysed and centrifuged. Subsequently, each sample was incubated with Protein A/G-agarose beads (Pierce) and primary antibodies (Supplementary Table 2). The immunocomplexes was washed by IP buffer for immunoblotting analysis with
Fig. 1. Effects of Trim69 on metabolic disorder and hippocampus injury in mice challenged with HFD. (A) RT-qPCR and (B,C) Western blot analysis of Trim69 expression in hippocampus of HFD-fed mice. (D) Immunofluorescent analysis of Trim69 and NeuN in hippocampus of HFD-challenged mice. (E) Body weight of mice after HFD treatment. Calculation of (F) fasting blood glucose levels and (G) insulin levels. (H) OGTT results were showed. (I) Nissl staining of CA3 and CA1 in hippocampus of mice. (J) TUNEL staining (up panel) and immunohistochemical analysis of Caspase-3 (down panel) in hippocampus of mice. Plots were mean ± SEM values. *p < 0.05 and **p < 0.01; þp < 0.05 vs HFD/WT group.
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the exhibited primary antibodies and corresponding secondary antibodies.
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two groups; one-way analysis of variance (ANOVA) was used for multiple comparisons with Bonferroni post hoc analysis. P < 0.05 was considered to be significant.
2.10. Ubiquitination assay BV2 cells were pre-treated with LPS (100 ng/ml) for 24 h and MG132 for 6 h. Endogenous ASK1 was immunoprecipitated from protein lysate using the ASK1 antibody and then immunoblotted with the anti-ubiquitin antibody to capture polyubiquitinated ASK1. BV2 cells were co-transfected with Myc-Ub, HA-ASK1 or Flag-Trim69 plasmids. 24 h after transfection, the cells were treated with LPS for further 24 h. Then, MG132 was added to the culture medium for 6 h prior to harvesting. Cells were split into two aliquots, one for immunoblotting and the other for an ubiquitination assay. For ubiquitination assay, the anti-HA antibody was used to immunoprecipitate ASK1 and then detected the ubiquitination using a Myc-tag antibody. 2.11. Flow cytometry analysis Annexin V-FITC apoptosis detection kit (Sigma Aldrich) was used following the manufactured protocols on a flow cytometer (Becton and Dickinson, USA) to analyze the populations of cells. 2.12. Statistical analysis All values were expressed as the mean ± SEM using GraphPad Prism 6.0 software (GraphPad Software, San Diego, USA). Statistical analysis was performed using the two-tailed Student's t-test for
3. Results 3.1. Effects of Trim69 on metabolic disorders and hippocampal injury in mice challenged with HFD To investigate the effects of Trim69 on metabolic-stress-induced hippocampal injury, the expression of Trim69 was measured. As shown in Fig. 1AeC, hippocampal Trim69 expression from mRNA and protein levels was markedly down-regulated in HFD-fed mice. Immunofluorescent analysis suggested that both Trim69 and NeuN expression levels were reduced by HFD challenge (Fig. 1D). Then, HFD-treated mice with or without Trim69 knockout were used to further explore the role of Trim69 in regulating hippocampal damage. As shown in Fig. 1E, HFD feeding led to a significant increase in the body weight of WT mice, which was more pronounced in Trim69-KO mice. This showed that HFD-induced elevation of fasting blood glucose and insulin levels was enhanced by the deficiency of Trim69 (Fig. 1F and G). OGTT analysis further indicated that Trim69 ablation markedly exacerbated HFD-induced insulin resistance in HFD-fed mice (Fig. 1H). Nissl staining showed that HFD treatment led to significant reduction in the number of survival neurons in CA3 and CA1 areas, which was further decreased by Trim69 deficiency (Fig. 1I). We also found the number of TUNELand caspase-3-positive cells in the hippocampi of mice to be aggravated in HFD-challenged mice with Trim69 deletion (Fig. 1J).
Fig. 2. Trim69 deficiency elevates inflammatory response in hippocampus of HFD-treated mice. (A) ELISA and (B) RT-qPCR analysis of TNF-a, IL-1b and IL-6 in serum and hippocampus tissues, respectively. (C) Western blot analysis of p-IkBa and p-NF-kB in hippocampus of mice. (D) Immunofluorescent analysis of p-NF-kB and Iba-1 in hippocampus of HFD-fed mice with or without Trim69 expression. (E) Western blot analysis of p-MKK4, p-MKK7 and p-JNK in hippocampus of mice. Plots were mean ± SEM values. *p < 0.05 and ** p < 0.01.
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These findings indicated that Trim69 played an essential role in metabolic stress-induced metabolic disorder and hippocampal damage.
3.2. Trim69 deficiency elevates inflammatory response in hippocampus of HFD-treated mice ELISA and RT-qPCR analysis indicated that HFD-enhanced systematic and hippocampal TNF-a, IL-1b, and IL-6 expression were further promoted in HFD-fed mice with Trim69 knockout (Fig. 2A and B). Western blot results indicated that the expression of phosphorylated IkBa and NF-kB in the hippocampus of HFD mice was markedly elevated by Trim69 deficiency (Fig. 2C). This indicated Trim69 expression plays a role in HFD-induced inflammatory response in the hippocampus of mice. Iba-1 is a hallmark of microglia activation, playing an essential role in regulating neuroinflammation, and this has been investigated [20,21]. Immunofluorescent analysis suggested that Trim69 ablation visibly upregulated the expression of p-NF-kB and Iba-1 in the hippocampus of HFD-fed mice over the HFD group (Fig. 2D). Given that Trim69 expression was correlated with JNK activation, we then investigated JNK signaling. As shown in Fig. 2E, the levels of expression of p-MKK4, p-MKK7, and p-JNK in hippocampi of mice were highly induced by HFD feeding, while being further promoted in Trim69-KO mice. These results suggested that Trim69-regulated hippocampal damage in HFD-fed mice was closely associated with the inflammatory response.
3.3. Trim69 directly interacts with ASK1 ASK1 was found to drive the inflammatory response in tissues mainly through a down-streaming JNK pathway. ASK1 activation was measured to further calculate the effects of Trim69 on HFDinduced hippocampal inflammation. As expected, Trim69 knockout markedly accelerated the expression of p-ASK1 in the hippocampus of HFD-fed mice (Fig. 3A). In this way, Trim69 showed a suppressive role in the ASK1 activation. We used microglial cells, which play a critical role in regulation of inflammation in the central nervous system, in subsequent studies [22]. We found that Trim69 inhibited ASK1 phosphorylation in a dose-dependent manner in LPS-incubated microglial cells transfected with varying amounts of Flag-Trim69 plasmids (Fig. 3B). Furthermore, specific interaction between Trim69 and ASK1 was observed by co-IP assays (Fig. 3C). ASK1 activation is associated with its polyubiquitination [23]. Within expectation, LPS stimulation significantly induced ASK1 ubiquitination and up-regulated ASK1 phosphorylation in BV2 cells (Fig. 3D). Over-expression of Trim69 in microglial cells led to substantial deubiquitination of ASK1 and suppression of its activation in response to LPS (Fig. 3E). 3.4. Trim69 expression protects against inflammation and apoptosis in microglia cells stimulated by LPS The expression of Trim69 was knocked down after infection with its targeting shRNAs (Fig. 4A). Also, the phosphorylation of ASK1 was inhibited by the suppression of Trim69 in microglial cells
Fig. 3. Trim69 directly interacts with ASK1. (A) Western blot analysis of p-ASK1 in hippocampus of mice from the indicated groups. (B) Western blot analysis of ASK1 and p-ASK1 in BV2 cells transfected with different amounts of Flag-tagged Trim69. (C) CO-IP results of BV2 cells transfected with Flag-tagged Trim69 or HA-ASK1. Flag and HA antibodies were applied for western blotting probes. (D) Western blot analysis of ASK1 ubiquitination and total ASK1 after IP for ASK1 (up panel), and p-ASK1 and ASK1 in lysate of BV2 cells stimulated with or without LPS (100 ng/ml) for 24 h. (E) Western blot analysis of ASK1 ubiquitination in BV2 cells with the transfection of plasmids as shown in the absence or presence of LPS (100 ng/ml) for 24 h. Plots were mean ± SEM values. *p < 0.05 and **p < 0.01.
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Fig. 4. Trim69 expression protects against inflammation and apoptosis in microglia cells stimulated by LPS. (A) Western blot analysis of Trim69 in BV2 cells transfected with different Trim69-targeting shRNAs (shTrim69) showing different inhibition efficacy. (B) Western blot analysis of p-ASK1 in BV2 cells transfected with different Trim69-targeting shRNAs with or without stimulation of LPS (100 ng/ml, 24 h). (C) Western blot analysis of p-ASK1, p-MKK4, p-MKK7, p-JNK and p-NF-kB in BV2 cells transfected with Trim69targeting shRNA with or without stimulation of LPS (100 ng/ml, 24 h). (D) RT-qPCR analysis of TNF-a, IL-1b and IL-6 and (E) flow cytometry analysis for apoptotic cell death in shTrim69-transfected BV2 cells with or without LPS stimulation (100 ng/ml) for 24 h. (F) Western blot analysis of p-ASK1, p-MKK4, p-MKK7, p-JNK and p-NF-kB in BV2 cells infected with Trim69-overexpressing Ad-Trim69 with or without stimulation of LPS (100 ng/ml, 24 h). (G) RT-qPCR analysis of TNF-a, IL-1b and IL-6 and (H) flow cytometry analysis for apoptotic cell death in BV2 cells infected with Trim69-overexpressing Ad-Trim69 in the absence or presence of LPS (100 ng/ml, 24 h). (I) Schematic of the role and regulatory mechanisms of Trim69 on HFD-induced hippocampus injury. Plots were mean ± SEM values. *p < 0.05 and **p < 0.01.
(Fig. 4B). Then, the shTrim69-3 was further applied to microglial cells for Trim69 inhibition. Western blot analysis suggested that the suppression of Trim69 further promoted the expression of p-ASK1, p-MKK4, p-MKK7, p-JNK, and p-NF-kB in BV2 cells upon LPS stimulation (Fig. 4C). Consistently, in LPS-incubated microglial cells, shTrim69 infection markedly increased the mRNA levels of TNF-a, IL-1b, and IL-6, as well as the rate of apoptotic cell death (Fig. 4D and E). In contrast, over-expressing Trim69 decreased the expression of p-ASK1, p-MKK4, p-MKK7, p-JNK, and p-NF-kB in microglial cells with LPS stimulation, along with subsequent down-regulation of TNF-a, IL-1b, and IL-6 by Western blot and RT-qPCR analysis (Fig. 4F and G). LPS-induced apoptosis in BV2 cells stimulated by LPS was visibly alleviated by Trim69 over-expression (Fig. 4H). These results indicated that Trim69 protected cells from inflammation and apoptosis in microglial cells stimulated with LPS. 4. Discussion In this study, we established the essential role of Trim69 in regulating the progression of hippocampal injury induced by HFD. We found Trim69 expression to be markedly up-regulated in the hippocampi of HFD-fed mice. Trim69 deficiency significantly accelerated HFD-induced metabolic disorder and hippocampal injury, which was closely associated with the progression of inflammation and apoptosis by activating NF-kB and JNK signaling
pathways. Trim69 expression negatively regulated ASK1 phosphorylation in the hippocampi of HFD-fed mice, and in LPSstimulated microglial cells. We verified that Trim69 directly interacted with and deubiquitinated ASK1 in microglial cells. The protective role of Trim69 against inflammation and apoptosis was further confirmed in LPS-incubated microglial cells. Our findings demonstrated that treatments promoting the deubiquitinating activity of Trim69 to inhibit ASK1 activation were promising leads as hippocampal injury therapies (Fig. 4I). The tripartite motif family proteins (TRIMs) share three conserved domains, an N-terminal Really Interesting New Gene (RING) domain, one or two B-Boxes (B1/B2), and a coiled-coil (CC) domain. These play crucial roles in various cellular processes, including apoptosis, neurogenesis, and innate immune responses [9,10]. Trim69, as a TRIM family member, is an IFN-inducible virus restriction factor that was found to be involved in apoptosis, tumor control, and zebrafish development [12,24,25]. Recently, TRIM69 was found to significantly suppress ultraviolet B (UVB)-induced apoptosis and oxidative stress. It could also interact with p53 and induce its ubiquitination, and functioned as an inhibitory factor during cataractogenesis [11]. Trim69 also regulates zebrafish brain development, which was largely associated with the activation of JNK signaling [12]. Inflammation and apoptosis are two critical pathologies that contribute to the progression of hippocampal injury associated with obesity [4,5,26,27]. In line with previous
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studies, we here found that HFD led to significant increases in the expression of pro-inflammatory cytokines, including TNF-a, IL-1b, and IL-6. This process took place largely by increasing the activation of IkBa/NF-kB signaling pathway. This is the first report to show that HFD-stimulated NF-kB activation was further promoted by the Trim69 deficiency. The JNK signaling pathway also plays a significant role in promoting the NF-kB signaling to promote the development of inflammatory response under diverse pathological processes [13,28]. JNK could be activated by MAPK kinases (MAPKKs), including mitogen-activated protein kinases 4 and 7 (MKK4 and MKK7) [29]. MAPKKs can in turn be activated by apoptosis signal regulating kinase group (ASK1), also known as MAP3K5 [30]. Of the MAPKKK molecules, ASK1 was found to be ubiquitously expressed and activates the MKK4/7-JNK signaling cascades [31]. ASK1 is a crucial facilitator and therapeutic target for preventing brain injury associated with obesity [32]. In our study, we found that HFD treatment markedly induced the phosphorylation of MKK4, MKK7, and JNK in the hippocampi of mice, and this can be further accelerated by Trim69 deletion, along with enhanced ASK1 phosphorylation (Fig. 4I). These results were consistent with the alterations of inflammatory response. IP analysis further demonstrated that Trim69 could directly interact with ASK1 and negatively regulate ASK1 activation. ASK1 polyubiquitination is related to its activation [33,34]. Our in vitro experiments suggested that LPS treatment of microglial cells led to significant ASK1 ubiquitination as well as upregulated levels of phosphorylated ASK1. Trim69 over-expression in microglial cells caused substantial ASK1 deubiquitination and suppression of its activation upon LPS stimulation. These findings further indicated that ASK1 modulation by Trim69 took place through activation-associated deubiquitination during the development of hippocampal injury induced by HFD. In conclusion, our findings here provide the first solid evidence that the polyubiquitination-dependent activation of ASK1 signaling pathway in response to LPS can be inhibited by Trim69. Furthermore, the Trim69/ASK1 pathway showed protective effects to prevent inflammation and apoptotic cell death, which might be involved in the treatment of hippocampal injury induced by obesity or associated metabolic stresses (Fig. 4I). However, further studies are still warranted to explore the effects of Trim69 on more specific mediators of hippocampal injury for developing effective treatments. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.027. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.05.027 References [1] M. Kivipelto, et al., Obesity and vascular risk factors at midlife and the risk of dementia and Alzheimer disease, Arch. Neurol. 62 (10) (2005) 1556e1560. [2] B.E. Levin, The obesity epidemic: metabolic imprinting on genetically susceptible neural circuits, Obes. Res. 8 (4) (2000) 342e347. [3] J.P. Thaler, et al., Obesity is associated with hypothalamic injury in rodents and humans, J. Clin. Investig. 122 (1) (2012) 153e162. [4] S.D. Bilbo, et al., Enduring consequences of maternal obesity for brain
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