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. 2020 Jun 5:13:100.
doi: 10.3389/fnmol.2020.00100. eCollection 2020.

Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery

Shen Zhao  1   2 , Peng Xiao  1   2 , Hao Cui  3   4 , Ping Gong  5 , Caijing Lin  1   2 , Feng Chen  1   2 , Ziren Tang  3   4
Affiliations

Hypothermia-Induced Ubiquitination of Voltage-Dependent Anion Channel 3 Protects BV2 Microglia Cells From Cytotoxicity Following Oxygen-Glucose Deprivation/Recovery

Shen Zhao et al. Front Mol Neurosci. .

Abstract

Background: Hypothermia attenuates microglial activation and exerts a potential neuroprotective effect against cerebral ischemic-reperfusion (I/R) injury. However, the underlying mechanism remains to be elucidated. In this in vitro study, a model of oxygen-glucose deprivation, followed by recovery (OGD/R), was used to investigate whether hypothermia exerts anti-inflammatory and anti-apoptosis properties via enhanced ubiquitination and down-regulation of voltage-dependent anion channel 3 (VDAC3) expression. Methods: BV2 microglia were cultured under OGD for 4 h following reperfusion with or without hypothermia for 2, 4, or 8 h. M1 and M2 microglia markers [inducible nitric oxide synthase (iNOS) and arginase (Arg)1] were detected using immunofluorescence. The levels of pro-inflammatory cytokines [tumor necrosis factor (TNF) α, interleukin (IL)-1β], and anti-inflammatory factor (IL-10) were determined using enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential (ΔΨm) was assayed by JC-1 staining using a flow cytometer. Expression of caspase-3, cleaved caspase-3, and VDAC3 were assessed using western blot analysis. The cellular locations and interactions of ubiquitin and VDAC3 were identified using double immunofluorescence staining and immunoprecipitation (IP) assay. Also, the level of the VDAC3 mRNA was determined using a quantitative polymerase chain reaction (qPCR). Results: Hypothermia inhibited the OGD/R-induced microglia activation and differentiation into the M1 type with pro-inflammatory effect, whereas it promoted differentiation to the M2 type with anti-inflammatory effect. Hypothermia attenuated OGD/R-induced loss of Δψm, as well as the expression of apoptosis-associated proteins. Compared to normothermia, hypothermia increased the level of ubiquitinated VDAC3 in the BV2 microglia at both 2 and 8 h of reperfusion. Furthermore, hypothermia did not attenuate VDAC3 mRNA expression in OGD/R-induced microglia. Conclusions: Hypothermia treatment during reperfusion, attenuated OGD/R-induced inflammation, and apoptosis in BV2 microglia. This might be due to the promotion of VDAC3 ubiquitination, identifying VDAC3 as a new target of hypothermia.

Keywords: BV2 microglial cells; VDAC3; cytotoxicity; hypothermia; oxygen-glucose deprivation/recovery.

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Figures

Figure 1
Figure 1
Effects of hypothermia on oxygen-glucose deprivation, followed by recovery (OGD/R)-induced cytotoxicity in microglia. Cell viability was determined using the cell counting kit-8 (CCK-8) assay. Values show the mean ± SD (n = 5). *P < 0.05, vs. Sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT group; ▵P < 0.05, vs. OGD/R4h-NT group; ▴P < 0.05, vs. OGD/R8h-NT group. White column = baseline; black solid column = OGD or OGD/R groups.
Figure 2
Figure 2
Effects of hypothermia on the immunoreactivity of anti-induced nitric oxide synthase (iNOS)-positive microglia using immunofluorescence assays. (A) Immunofluorescence images showing the BV2 microglia following OGD/R labeled with the iNOS antibody or secondary antibody (control negative). Red fluorescence indicates iNOS-positive cells, while blue fluorescence indicates 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)-labeled nuclei. Scale bar: 20 μm. (B) Dot plots (upper panel) and a bar graph (lower panel) showing a quantitative analysis of red/blue fluorescence ratios (25 random observations for each group). Values show the mean ± SD, n = 5. *P < 0.05, vs. sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT groups; ▴P < 0.05, vs. OGD/R8h-NT groups.
Figure 3
Figure 3
Effects of hypothermia on the immunoreactivity of arginase 1 (Arg1)-positive microglia using immunofluorescence assays. (A) Immunofluorescence images showing the BV2 microglia following OGD/R labeled with the Arg1 antibody or secondary antibody (control negative). Green fluorescence indicates Arg1-positive cells, while blue fluorescence indicates DAPI-labeled nuclei. Scale bar: 20 μm. (B) Dot plots (upper panel) and a bar graph (lower panel) showing a quantitative analysis of green/blue fluorescence ratios (25 random observations for each group). Values show the mean ± SD, n = 5. *P < 0.05, vs. sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT groups; ▴P < 0.05, vs. OGD/R8h-NT groups.
Figure 4
Figure 4
Effects of hypothermia on pro-inflammatory and anti-inflammatory cytokine release in OGD/R-induced microglia. Protein levels of interleukin (IL)-1β (A) tumor necrosis factor (TNF)-α (B) and IL-10 (C) in culture media were detected using enzyme-linked immunosorbent assay (ELISA). Values show the mean ± SD, n = 5. *P < 0.05, vs. sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT groups; ▵P < 0.05, vs. OGD/R4h-NT groups; ▴P < 0.05, vs. OGD/R8h-NT groups.
Figure 5
Figure 5
Effect of hypothermia on the loss of mitochondrial membrane potential (Δψm) in microglial cells. (A) Scatter diagram of5,5’, 6,6’-tetrachloro-1,1’, 3,3’-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1) dye from different groups. a. Sham; b. OGD; c. OGD/R2h-NT; d. OGD/R4h-NT; e. OGD/R8h-NT; f. OGD/R2h-HT; g. OGD/R4h-HT; h. OGD/R8h-HT. (B) Bar graph showing the fluorescence ratio R2/R1 in the different groups. Values show the mean ± SD (n = 5). *P < 0.05, vs. Sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT groups; ▵P < 0.05, vs. OGD/R4h-NT groups; ▴P < 0.05, vs. OGD/R8h-NT groups.
Figure 6
Figure 6
Effect of hypothermia on the expression of apoptosis-associated proteins in microglial cells. (A) Representative western blot images showing the expression of caspase3, cleaved caspase3, and voltage-dependent anion channel 3 (VDAC3) in different groups. β-Actin was used as an internal control. (B) Bar graph showing the quantification of capase3/β-actin in different groups. (C) Bar graph showing the quantification of cleaved capase3/β-actin in different groups. (D) Bar graph showing the quantification of cleaved VDAC3/β-actin in different groups. Values show the mean ± SD (n = 5). *P < 0.05, vs. Sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT groups; ▵P < 0.05, vs. OGD/R4h-NT groups; ▴P < 0.05, vs. OGD/R8h-NT groups.
Figure 7
Figure 7
Effects of hypothermia on the immunoreactivity of ubiquitin-positive and anti-VDAC3-positive microglia using double immunofluorescence assays. (A) Immunofluorescence images showing the BV2 microglia following OGD/R labeled with ubiquitin (green) and VDAC3 (red) antibody or secondary antibodies (control negative). Blue fluorescence indicates DAPI-labeled nuclei. Scale bar: 20 μm. (B) Quantification of the colocalization coefficient between ubiquitin and VDAC3. Values show the mean ± SD, n = 5. *P < 0.05, vs. sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT groups; ▴P < 0.05, vs. OGD/R8h-NT groups.
Figure 8
Figure 8
Hypothermia induced ubiquitination of VDAC3 in microglial cells. (A) To detect non-specific binding with magnetic beads, OGD/R8h-HT cells were immunoprecipitated with IgG or anti-ubiquitin, followed by western blotting (WB) with anti-VDAC3 and anti-ubiquitin antibodies. Anti-ubiquitin and anti-VDAC3 were used for WB to determine the ubiquitination level of VDAC3 (left). WB data of ubiquitin, VDAC3, and β-actin from the input (right). (B) Immunoblot analyses of ubiquitinated VDAC3 in different groups. Cell lysates were immunoprecipitated with anti-ubiquitin antibody, followed by WB with the anti-ubiquitin antibody and VDAC3 antibodies. β-Actin was used as an internal control of input. (C) Quantification of ubiquitinated VDAC3 in experimental groups/sham group (Left) and ubiquitinated proteins/β-actin in different groups (right). Results are shown as mean ± SD (n = 5). Left: *P < 0.05, vs. Sham group; θP < 0.05, vs. OGD group; φP < 0.05, vs. OGD/R2h-HT groups; #P < 0.05, vs. OGD/R2h-NT groups; ▴P < 0.05, vs. OGD/R8h-NT groups.
Figure 9
Figure 9
Effect of hypothermia on VDAC3 mRNA expression in microglial cells. The normalized VDAC3 mRNA level was determined in each group using real-time RT-PCR. *P < 0.05, vs. sham group. n = 5 repeated from five independent experiments.
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