Peli1 Contributions in Microglial Activation, Neuroinflammatory Responses and Neurological Deficits Following Experimental Subarachnoid Hemorrhage

Abstract

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is closely associated with neuroinflammation. Microglial activation is an early event that leads to neuroinflammation after SAH. Peli1 is an E3 ubiquitin ligase that mediates the induction of pro-inflammatory cytokines in microglia. Here we report Peli1 contributions in SAH mediated brain pathology. An SAH model was induced by endovascular perforation in adult male C57BL/6J mice. Peli1 was markedly induced in mice brains in a time-dependent manner and was predominantly expressed in CD16/32-positive microglia after SAH. Using genetic approaches, we demonstrated that decreased Peli1 significantly improved neurological deficits, attenuated brain edema, reduced over-expression of pro-inflammatory cytokine IL-6 and modified apoptotic/antiapoptotic biomarkers. In addition, Peli1 downregulation suppressed ERK and JNK phosphorylation levels via the downregulation of cIAP1/2 expression, subsequently reducing inducible nitric oxide synthase (iNOS) expression after SAH. Therefore, these findings demonstrate that Peli1 contributes to microglia-mediated neuroinflammation in EBI by mediating cIAP1/2 activation, thus promoting the activation of MyD88-dependent MAPK pathway after experimental SAH. Our findings also showed that Peli1 could promote the expression of M1 microglia polarization biomarker CD16/32 and iNOS after SAH. Targeting Peli1 exerts neuroprotective effects during EBI after SAH, thus could provide potential option for prevention-therapy in high-risk individuals.

Keywords: Peli1; early brain injury; microglia; neuroinflammation; subarachnoid hemorrhage.

Figure 1

Peli1 is upregulated and mediates microglial activation in subarachnoid hemorrhage (SAH) model. (A) A mass of blood clots were particularly pronounced around arterial circle of Willis after SAH. (B) Hematoxylin & eosin (H&E) staining of coronal section from mice subjected to sham and SAH on 48 h for visualizing myeloid cells infiltration in subarachnoid layers (arrows). Original magnification is ×400. Scale bars are 50μm. (C) Western blot showing higher levels of Peli1 protein expression at 6, 24, 48 and 72 h after SAH (left). Quantification (right) of normalized Peli1 levels were shown (n = 3 per time point, *P = 0.009, **P < 0.001, ^#P < 0.001, ^##P < 0.001 vs. Sham). (D) Left: Western blot showing TLR4 and cIAP1/2 expression at the indicated time points after SAH. Right: quantification of normalized TLR4 and cIAP1/2 levels (n = 3 per time point, *P < 0.01, ^#P < 0.001, ns indicating non-significant, vs. Sham group). (E) Immunofluorescent staining for CD16/32 (red) reflecting co-localization of Peli1 (green) by activated microglia in mouse brains 48 h after SAH and sham-operated group. Nuclei were stained blue by DAPI. Original magnification is ×400. Scale bars are 50 μm. The experiments were repeated three times.

Figure 2

Knockdown of Peli1 alleviates neurological deficits and reduces T2 hyperintensity after SAH. (A) Similar SAH grades were observed in the negative control (NC) lentivirus-treated (Peli1-NC) group and Peli1-knockdown lentivirus- treated (Peli1-KD) group at 48 h after SAH (n = 6 per group). (B) The neurological scores were significantly lower in Peli1-NC group than that of sham group 48 h after SAH (n = 6 per group, *P < 0.001). Downregulation of Peli1 significantly improved neurologic scores compared with Peli1-NC group 48 h after SAH (n = 6 per group, ^#P < 0.001, vs. Peli1-NC group). (C) Coronal T2 MRI images of sham and SAH mice brains at 48 h. T2-hyperintensity was observed with markedly increased in mice undergoing endovascular perforation compared with sham-operated group. The white arrows indicate the white matter regions. (D) The measured volume of T2-hyperintense region showed an increase in Peli1-NC group of animals 48 h after SAH in comparison to sham-operated animals (n = 3 per group, *P < 0.001). Knockdown of Peli1 significantly attenuated T2 hyperintensity compared with Peli1-NC group of animals 48 h after SAH (n = 3 per group, ^#P < 0.001).

Figure 3

Downregulation of Peli1 attenuates apoptosis and production of pro-inflammatory cytokines after SAH. (A) Immunoblot analysis of Peli1 expression in sham, Peli1-NC and Peli1-KD groups of mice. (B) Quantification of Peli1 expression normalized to GAPDH showed that the levels of Peli1 protein were increased in Peli1-NC mice 48 h after SAH compared to sham group (n = 5 per group, *P < 0.001). However, administration of Peli1-KD lentiviral vectors significantly attenuated expression of Peli1 compared to Peli1-NC group 48 h after SAH (^#P = 0.016). (C–E) Immunoblotting of apoptosis-related protein Bax and Bcl2 in the brain extracts of sham-operated mice, Peli1-NC and Peli1-KD mice 48 h after SAH. Downregulation of Peli1 significantly reduced pro-apoptotic protein Bax, and enhanced the decreased level of Bcl2. The normalized Bax expression were shown graphically in (D) (n = 5 per group, *P < 0.001 vs. sham; ^#P < 0.001 vs. SAH + Peli1-NC mice). Quantifications of the normalized Bcl2 protein levels were shown in (E) (n = 5 per group, *P < 0.001 vs. sham; ^#P = 0.001 vs. SAH + Peli1-NC mice). (F) ELISA analysis showing that downregulation of Peli1 markedly alleviated the expression of IL-6 48 h after SAH (n = 3, *P < 0.001 vs. sham; ^#P = 0.005 vs. SAH + Peli1-NC mice).

Figure 4

Downregulation of Peli1 inhibits inducible nitric oxide synthase (iNOS) expression and suppresses activation of MAPK signaling pathway. (A) Immunoblotting of iNOS was performed in brain extracts from sham- or SAH-operated Peli1-NC and Peli1-KD mice. Downregulation of Peli1 reduced the expression of iNOS 48 h after SAH. Quantification of iNOS normalized to GAPDH expression was shown in (B) (n = 5 per group, *P < 0.001 vs. sham; ^#P = 0.002 vs. SAH + Peli1-NC group). (C–E) Peli1 activates MAPK signaling pathway. Western blot analysis of phosphorylated ERK and JNK in brain homogenates from sham- or SAH-operated Peli1-NC and Peli1-KD mice. Downregulation of Peli1 decreased phosphorylation of JNK and ERK 48 h after SAH. Quantification of normalized p-JNK was shown in (D) (n = 5 per group, *P < 0.001 vs. sham; ^#P = 0.002 vs. SAH + Peli1-NC group). Quantification of normalized p-ERK was shown in (E) (n = 5 per group, *P < 0.001 vs. sham; ^#P = 0.013 vs. SAH + Peli1-NC group).

Figure 5

The Effects of Peli1 knockdown on cIAP1/2 expression. (A) Immunoblotting of cIAP1/2 expression in sham, Peli1-NC and Peli1-KD groups of mice. (B) Quantification of cIAP1/2 normalized to GAPDH expression showed that Peli1 enhanced cIAP1/2 expression 48 h after SAH (n = 5 per group, *P < 0.001 vs. sham; ^#P < 0.001 vs. SAH + Peli1-NC group). (C–E) Downregulation of Peli1 has no effects on protein level of TLR4 and MyD88 48 h after SAH. Quantification of TLR4 normalized to GAPDH expression was shown in (D) (n = 5 per group, *P = 0.004 vs. sham; ns, P = 0.891, indicating non-significant, vs. SAH + Peli1-NC group). The normalized MyD88 was shown in (E) (n = 5 per group, *P = 0.006 vs. sham; ns, P = 0.771, indicating non-significant, vs. SAH + Peli1-NC group).

Figure 6

Schema depicting the Peli1-mediated pathway following SAH. TLR4 signaling is initiated by SAH stimuli. Increased Peli1 after SAH positively regulates MyD88-dependent TLR4 signaling via cIAP1/2 activation. Upregulated cIAP1/2 facilitates phosphorylation of MAPK signaling pathway and activates pro-apoptotic pathway. These combined effects promotes microglia polarization into the M1 phenotype, sequentially releasing destructive pro-inflammatory cytokines to amplify the inflammatory response in the central nervous system (CNS). Suppression of Peli1 by Peli1-shRNA lentivirus impairs the induction of pro-inflammatory cytokines.