Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury

Abstract

Background: Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI.

Methods: SH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett's T3 test (for unequal homogeneity) were used for statistical tests.

Results: ERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1^S616E or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed.

Conclusion: Inhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model. Video Abstract.

Keywords: Autophagy; CIRI; Drp1; ERK; Mfn2; Mitochondrial dynamics; OGD/R.

Fig. 1

Inhibition of ERK protects SH-SY5Y cells from OGD/R-induced injury

Fig. 2

Downregulation of autophagy by ERK inhibition alleviates OGD/R-induced injury and death in SH-SY5Y cells

Fig. 3

Transfection efficiency of si-RNA and overexpression plasmid. A Representative immunoblot bands of p-Drp1 and Drp1; B, C, Quantitative analysis of p-Drp1 and Drp1; D, SH-SY5Y cells transfected with Drp1^S616E-GFP-M90 plasmid after 24 h observed under fluorescence microscope; E, Quantitative analysis of fluorescence intensity; F, H, Representative immunoblot bands of Mfn2 after transfected with si-Mfn2 and Mfn2 pcDNA3.1; G, I, Quantitative analysis of Mfn2. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. si-NC: Drp1 si-RNA negative control group; si-751/si-843/si1684: Drp1 si-RNA sequences; Drp1^S616E: Drp1^S616E-GFP-M90 plasmid; Vector: Drp1^S616E negative control group; si-1/si-2/si-3: Mfn2 si-RNA sequences; OE-Mfn2: Mfn2 pcDNA3.1. *P < 0.05 vs. si-NC and Control; #P < 0.05 vs. Vector

Fig. 4

Inhibition of ERK alleviates p-Drp1S616 dependent mitochondrial fission and injury after OGD/R in SH-SY5Y cells. A Representative immunoblot bands of p-Drp1 and Drp1; B, C, Quantitative analysis of p-Drp1 and Drp1; D, Transmission electron microscopy images of mitochondria, the red arrows shows the mitochondria; E, Quantitative analysis of mitochondrial area. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. si-NC: Drp1 si-RNA negative control group; si-751/si-843/si1684: Drp1 si-RNA sequences; Drp1^S616E: Drp1^S616E-GFP-M90 plasmid; Vector: Drp1^S616E negative control group. *P < 0.05 vs. Control, #P < 0.05 vs. si-NC, &P < 0.05 vs. si-843, $P < 0.05 vs. si-1684, @P < 0.05 vs. PD + si-NC

Fig. 5

Inhibition of ERK downregulates autophagy by reducing Drp1-dependent mitochondrial fission after OGD/R in SH-SY5Y cells. A, E, Representative immunoblot bands of LC3B, Beclin1 and p62; B-D, F-H, Quantitative analysis of LC3B, Beclin1 and p62; I. Transmission electron microscopy images of autophagosome/autolysosome (red arrows). All data in this figure are representative of three independent repeats and are shown as mean ±SEM. si-NC: Drp1 si-RNA negative control group; si-751/si-843/si1684: Drp1 si-RNA sequences; Drp1^S616E: Drp1^S616E-GFP-M90 plasmid; Vector: Drp1^S616E negative control group. *P < 0.05 vs. Control, #P < 0.05 vs. si-NC, &P < 0.05 vs. si-843, $P < 0.05 vs. si-1684, @P < 0.05 vs. PD + si-NC

Fig. 6

Inhibition of ERK prevents mPTP opening and protects from OGD/R injury by reducing Drp1 activation in SH-SY5Y cells. A The opening of mPTP was detected by loading with calcein AM (green fluorescence). The intensity of green fluorescence was negatively correlated with the opening of mPTP, scare bar: 100 μm. B Quantitative analysis of calcein AM florescence intensity. C, D, LDH and CCK-8 assays were performed to detect cell injury and viability. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. si-NC: Drp1 si-RNA negative control group; si-Drp1: Drp1 si-RNA sequences; Drp1^S616E: Drp1^S616E-GFP-M90 plasmid; Vector: Drp1^S616E negative control group. *P < 0.05 vs. Control, #P < 0.05 vs. Model, &P < 0.05 vs. si-NC, $P < 0.05 vs. Vector

Fig. 7

p-ERK, p-Drp1 and LC3II are co-expressed in OGD/R-treated SH-SY5Y cells. A Multiple immunofluorescence images of p-ERK (green), p-Drp1 (pink) and LC3II (red) proteins at 24 h after OGD/R. Nuclei were stained with Dapi (blue), scare bar: 100 μm. Detail represents the enlarged view of the rectangles in Merge. B Quantitative analysis of p-ERK + p-Drp1 + LC3II positive cell rate. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. PD: PD98059 group; si-Drp1: Drp1 si-RNA sequences; Drp1^S616E: Drp1^S616E-GFP-M90 plasmid; *P < 0.05 vs. Control, #P < 0.05 vs. Model, &P < 0.05 vs. Drp1^S616E

Fig. 8

The expression of p-ERK, p-Drp1 and LC3II at different time points of OGD/R. A Multiple immunofluorescence images of p-ERK (green), p-Drp1 (pink) and LC3II (red) proteins at OGD1h/2 h/3 h reoxygenation 24 h, and OGD2h reoxygenation 12 h/24 hrs/36 hrs. Nuclei were stained with Dapi (blue), scare bar: 50 μm. Detail represents the enlarged view of the rectangles in Merge. B-D, Quantitative analysis of p-ERK, p-Drp1 and LC3II expression. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. OGD1h/2 h/3 h: oxygen-glucose deprivation 1 h/2 hrs/3 hrs and reoxygenation 24 h; R12h/24 h/36 h: oxygen-glucose deprivation 2 h and reoxygenation 12 h/24 hrs/36 hrs. *P < 0.05 vs. Control

Fig. 9

Inhibition of ERK promotes expression of mitochondrial fusion proteins by increment of Mfn2 after OGD/R in SH-SY5Y cells. A Representative immunoblot bands of mitochondrial fusion proteins: Opa1, Mfn2 and Mfn1; B-D, Quantitative analysis of expression of Opa1, Mfn2 and Mfn1. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. si-Mfn2(C): Mfn2 si-RNA without OGD/R group; PD: PD98059 group; Mfn2: Mfn2 si-RNA group. *P < 0.05 vs. Control, #P < 0.05 vs. Model, &P < 0.05 vs. PD

Fig. 10

Inhibition of ERK downregulates autophagy through increasing Mfn2 in OGD/R-treated SH-SY5Y cells. A Representative immunoblot bands of LC3B, Beclin1 and p62. B-D, Quantitative analysis of expression of LC3B, Beclin1 and p62. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. E The transmission electron microscopy images of autophagosomes/autolysosomes (red arrows). Scare bar: 1 μm. si-Mfn2(C): Mfn2 si-RNA without OGD/R group; PD: PD98059 group; Mfn2: Mfn2 si-RNA group. *P < 0.05 vs. Control, #P < 0.05 vs. Model, &P < 0.05 vs. PD

Fig. 11

Inhibition of ERK prevents mPTP opening and protects from OGD/R injury by increment of Mfn2 in SH-SY5Y cells. A, The opening of mPTP was detected by loading with calcein AM (green fluorescence). The intensity of green fluorescence was negatively correlated with the opening of mPTP, scare bar: 200 μm. B, Quantitative analysis of calcein AM florescence intensity. C, D, LDH and CCK-8 assays were performed to detect cell injury and viability. All data in this figure are representative of three independent repeats and are shown as mean ±SEM. si-Mfn2(C): Mfn2 si-RNA without OGD/R group; PD: PD98059 group; Mfn2: Mfn2 si-RNA group. *P < 0.05 vs. Control, #P < 0.05 vs. Model, %P < 0.05 vs. PD + Mfn2

Fig. 12

The schematic of hypothesis for ERK-mitochondrial fragmentation-autophagy axis-mediated OGD/R-induced injury in SH-SY5Y cells. Inhibition of ERK downregulates autophagy via ameliorating Drp1/Mfn2-dependent mitochondrial fragmentation, recovering mitochondrial function and reducing neuron death. OGD/R: Oxygen glucose deprivation/reoxygenation; p: Phosphorylation; ERK: Extracellular signal-regulated kinase; Drp1: Dynamin-related protein 1: Mfn2: Mitofusin2; LC3: Microtubule-associated protein light chain 3; Beclin1: BECN 1; p62: Sequestosome 1; mPTP: Mitochondrial permeability transition pore