Sirtuin 3 promotes microglia migration by upregulating CX3CR1

Abstract

We studied the role of Sirtuin 3 (SIRT3) in microglial cell migration in ischemic stroke. We used a middle cerebral artery occlusion (MCAO) model of focal ischemia. We then applied lentivirus-packaged SIRT3 overexpression and knock down in microglial N9 cells to investigate the underlying mechanism driving microglial cell migration. More microglial cells appeared in the ischemic lesion side after MCAO. The levels of SIRT3 were increased in macrophages, the main source of microglia, after ischemia. CX3CR1 levels were increased with SIRT3 overexpression. SIRT3 promoted microglial N9 cells migration by upregulating CX3CR1 in both normal and glucose deprived culture media. These effects were G protein-dependent. Our study for the first time shows that SIRT3 promotes microglia migration by upregulating CX3CR1.

Keywords: CX3CR1; Ischemia; SIRT3; microglia migration.

Figure 1.

Microglial cells were increased after ischemic stroke. (a) Representative images showed that microglia increased in the ipsilateral side after MCAO. (b) Semi-quantification analysis of the number of Iba-1 positive cells. Data were presented as mean± S.E.M., *p < 0.05, **p < 0.01. Five random fields were counted for each sample, n = 5 per group.

Figure 2.

SIRT3 levels were increased in macrophages after MCAO (a) and (b) CD11b+ F4/80+ are used for gating macrophage cells. (c) Flow cytometry analysis is performed to measure SIRT3 levels in macrophage cells. (d) Bar graph demonstrates relative SIRT3 levels in macrophage cells were increased after MCAO. Data were presented as mean± S.E.M., **p < 0.01 compared with Sham group, n = 5 per group.

Figure 3.

SIRT3 promoted the migratory ability of microglia. (a) Representative images illustrated that SIRT3 was overexpressed or knocked down in N9 cells. All Lenti-Virus vector containing the sequence of eGFP were visualized. Scale bars = 100 μm. (b)Schematic graph showed the plate structure for the migration assay. (c) Representative images showed that the migration of N9 cells transfected with Vector, shRNA and SIRT3 in normal or GD media. The migration of SIRT3 N9 cells increased compared with Vector N9 cells, and the migration of shRNA N9 cells decreased compared with Vector N9 cells both under normal or GD condition. Scale bars = 100 μm. Five random fields were counted for each group. Each experimental group was repeated three times. (d) Bar graph showed per field migrated cell numbers of N9 cells transfected with Vector, shRNA and SIRT3 in normal or GD media. Data were presented as mean± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 4.

SIRT3 regulated microglial cell migration via G protein-dependent CX3CR1. (a) Representative western blots of CX3CR1 were shown in vectors or SIRT3 transfected cells. CX3CR1 levels increased in SIRT3 N9 cells compared with Vector N9 cells. n = 5 per group. (b) Bar graph showed that the density of CX3CR1 in N9 cells transfected by Vector, shRNA or SIRT3 Lenti-virus. β-actin was an internal control. (c) Representative images illustrated the migration of Vector N9 cells after adding PTx to the culture medium. PTx can block the migration of N9 cells by administrated on upper or lower chamber. Scale bars = 100 μm. Five random fields were counted for each group. Each experimental group was repeated three times. (d) Bar graph showed the migrated cell numbers per field with and without PTx. (e) Representative images showed that migration of SIRT3+ N9 cells was blocked by PTx in both the normal and GD media. Scale bars = 100 μm. Five random fields were counted for each group. Each experimental group was repeated three times. (f) Bar graph showed the migrated SIRT3+ N9 cell numbers per field after adding PTx in the normal and GD media. Data were presented as mean± S.E.M., *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.