Overexpression of circDYM meliorated depressive-like behaviors induced by LPS
As described in our previous studies (28), the expression of circDYM in the hippocampus of mice were markedly decreased induced by LPS. Based on this point, we sought to verify the contribution of circDYM in the hippocampus of C57BL/6J mice by microinjecting either the circDYM-GFP lentivirus or the circControl-GFP lentivirus (Fig. 1A). As shown in Fig. 1B, mice treated with LPS preferred sucrose less compared to the control group and were significantly meliorated by circDYM overexpression. To further assess the impact of circDYM, two additional behavioral tests were used. In TST (Fig. 1C) and FST (Fig. 1D), immobility time was significantly longer in LPS group than in control group, which was markedly ameliorated in circDYM lentivirus-microinjected gruop.
Overexpression of circDYM inhibits autophagy and apoptosis induced by LPS
Although circDYM has been reported in our previous studies (28), the role it plays in microglia autophagy and survival induced by LPS has remained obfuscated. We explored the impact of LPS on mouse hippocampal microglia to deeply explore how LPS impacts autophagy and apoptosis of microglia. As shown in Fig. 1E, the expression of LC3B was markedly increased treated with LPS, and the increasing was ameliorated by circDYM. Furthermore, LPS-mediated upregulation of cleaved caspase-3 was ameliorated in mice of overexpression of circDYM compared with circCon group in the hippocampus (Fig. 1F). Moreover, circDYM significantly ameliorated the increase of LC3B and cleaved caspase-3 expression treated with LPS in BV2 cell (Fig. 1G-1H).
CircDYM bound to CEBPB
Further studies were required to elucidate the precise mechanisms of depression since it proved that circDYM plays a crucial role in depression. Then, we used the catRAPID algorithm to explore the interaction of circDYM with CEBPB and found two regions of CEBPB are predicted to have high interaction capacity with circDYM: 45–96 and 145–202 (Fig. 2A). CircDYM and CEBPB binding was determined by RIP assay (Fig. 2B). Furthermore, we created a circDYM mutant plasmid (45–96, 145–202) wherein the circDYM binding region that interacts with CEBPB was deleted. Compared to the mutant variant of circDYM, the unmodified circDYM showed stronger binding with CEBPB (Fig. 2C). As shown in Fig. 2D, there was colocalization of circDYM and CEBPB.
Overexpression of CEBPB increased autophagy and apoptosis induced by LPS
After it was established that LPS induces autophagy and apoptosis in microglia, further research was performed to clarify the vital role that CEBPB plays in the autophagy and apoptosis that LPS induces utilizing the CEBPB-ACT. In Fig. 2E and 2F, pretreatment of cells with CEBPB-ACT increased autophagy and apoptosis induced by LPS with evidencing by increased levels of LC3B-II and cleaved caspase-3, demonstrating that CEBPB is upstream of autophagy and apoptosis in LPS-treated BV2 cells.
Redistribution of CEBPB after LPS treatment
Furthermore, we seek to investigate the CEBPB expression in BV2 cells induced by LPS. CEBPB expression was notably increase in the nucleus in LPS group (Fig. 2G). These facts lead us to conclude that CEBPB was transferred from the cytoplasm to the nucleus. As shown in Fig. 2H, overexpression of circDYM decreased redistribution of CEBPB from the cytoplasm to the nucleus.
CEBPB regulates the expression of ZC3H4
Having determined that circDYM regulates CEBPB expression and redistribution, we investigated the role of CEBPB in BV2 cells treated with LPS. It seems clear that transfection with the CEBPB-ACT upregulated the expression of CEBPB in BV2 cells (Fig. 3A). Then, transfection with CEBPB-ACT significantly exacerbated the LPS-induced diminution of ZC3H4 expression (Fig. 3B). Contrarily, as shown in Fig. 3C, transfection with the CEBPB-NIC downregulated the expression of CEBPB in BV2 cells. The LPS-induced reduction of ZC3H4 expression was significantly improved after CEBPB-NIC transfection (Fig. 3D). Next, an in vivo experiment showed that mice treated with LPS exhibited significantly decreased expression of ZC3H4 compared with the control group (Fig. 3E) and these effects were significantly attenuated by circDYM (Fig. 3F). As shown in Fig. 3G, treatment of BV2 cells with LPS resulted in increased ZC3H4 in a time-dependent manner, with the peak activation at 12h and a gradual decrease thereafter. In line with these findings, circDYM lentivirus transfection significantly attenuated the LPS-induced decrease of ZC3H4 expression (Fig. 3H). To prove that circDYM is a CEBPB mediator, we co-transfected BV2 cells with CEBPB-ACT and circDYM. CircDYM overexpression enhanced ZC3H4 expression, and this change was significantly attenuated by circDYM (Fig. 3I).
Role of autophagy in the LPS-induced apoptosis
As shown in Fig. 4A-4B, BECN1 expression was elevated and LC3B-II production was enhanced LPS-treated BV2 cells. For the autophagy, P62 functions as a connector that links LC3B with ubiquitin moieties on misfolded proteins. Therefore, autophagy facilitates the elimination of P62 and ubiquitinated proteins. Consistently, P62 expression was downregulated in BV2 cells after treatment with LPS. We next sought to explore the apoptosis in the LPS-treated BV2 cells. As shown in Fig. 4C-4D, LPS increased the expression of Bcl-xl and decreased the expression of cleaved caspase-3. Having determined that LPS induced apoptosis, we next examined viability of LPS-treated cells. As shown in Fig. 4E, exposure to LPS reduced microglial survival. Moreover, cleaved caspase-3 was enhanced by the autophagy inducer rapamycin (Fig. 4F). To further confirm the role of autophagy in LPS-induced apoptosis, BV2 cells were treated with 3-MA, which decreased the level of cleaved caspase-3 (Fig. 4G).
Role of autophagy in LPS-induced viability in vitro
Having determined that circDYM regulates ZC3H4 expression, we next studied the role of ZC3H4 in apoptosis induced by LPS. As shown in Fig. 5A-5B, transfection with the ZC3H4-ACT upregulated the expression of ZC3H4 in BV2 cells. Moreover, transfection with ZC3H4-ACT contributed to significant amelioration of the increased expression of LC3B-II and cleaved caspase-3. Meanwhile, transfection with the ZC3H4-NIC downregulated the expression of ZC3H4 in BV2 cell and lead to significant aggravation of the increased expression of LC3B-II and cleaved caspase-3 (Fig. 5C-5D). To study the effects of ZC3H4 on viability, BV2 cells were transfected with ZC3H4-ACT and ZC3H4-NIC. As shown in Fig. 5E, ZC3H4-ACT ameliorated the decrease of cell viability caused by LPS compared with the control, while ZC3H4-NIC aggravated it (Fig. 5F).
Role of autophagy in LPS-induced viability in vivo
After demonstrating that ZC3H4, involved in regulation of autophagy, plays a critical role in microglia viability, we aimed to validate the role of microglial ZC3H4 in vivo. As shown in Fig. 6A-6B, the ZC3H4f/fIba-1Cre mice exhibited significantly lower expression of ZC3H4 compared with ZC3H4f/f mice. Next, the expression of LC3B-II and cleaved caspase-3 was reduced in ZC3H4f/fIba-1Cre mice administered LPS, demonstrating that ZC3H4 plays a vital role in microglia viability in vivo (Fig. 6C-6D).