Growth Differentiation Factor 15 Regulates Oxidative Stress-Dependent Ferroptosis Post Spinal Cord Injury by Stabilizing the p62-Keap1-Nrf2 Signaling Pathway

Abstract

Background: Spinal cord injury (SCI) is a severe traumatic disorder of the central nervous system (CNS) that causes irreversible damage to the nervous tissue. The consequent hemorrhage contributed by trauma induces neuronal ferroptosis post SCI, which is an important death mode to mediate neuronal loss. Growth differentiation factor 15 (GDF15) is a cytokine that regulates cell proliferation, differentiation, and death. However, the specific role of GDF15 in neuronal ferroptosis post SCI remains unknown.

Materials and methods: Neuronal ferroptosis in vitro was measured by detection of lipid peroxidation, glutathione, iron content, and reactive oxidative stress. In vivo, western blotting and immunofluorescence (IF) staining was utilized to measure ferroptosis post SCI. IF staining, TUNEL staining, hematoxylin-eosin staining, and Nissl staining were used to measure neurological damage. Finally, locomotor function recovery was analyzed using the Basso Mouse Scale and Louisville Swim Scale.

Results: GDF15 was significantly increased in neuronal ferroptosis and silencing GDF15 aggravated ferroptosis both in vitro and in vivo. Besides, GDF15-mediated inhibition of neuronal ferroptosis is through p62-dependent Keap1-Nrf2 pathway. In SCI mice, knockdown of GDF15 significantly exacerbated neuronal death, interfered with axon regeneration and remyelination, aggravated ferroptosis-mediated neuroinflammation, and restrained locomotor recovery.

Conclusion: GDF15 effectively alleviated neuronal ferroptosis post SCI via the p62-Keap1-Nrf2 signaling pathway and promoted locomotor recovery of SCI mice, which is suggested as a potential target on SCI pathogenesis and treatment.

Keywords: GDF15; ferroptosis; oxidative stress; p62-Keap1-Nrf2 pathway; spinal cord injury.

FIGURE 1

GDF15 was increased in SCI and neuronal ferroptosis. (A) Cell viability was detected by CCK-8 (n = 6). (B) Relative mRNA level of GDF15 in the spinal cord within a week post-injury (n = 6). (C) Western blotting of GDF15 protein levels in the spinal cord within a week post-injury (n = 6). (D) Bar graph showing a quantitative analysis of GDF15 expression (n = 6). (E) Western blotting of GDF15 and ferroptosis-associated proteins including ACSL4, FTH1, and GPX4 in Hemin-stimulated primary neurons within 24 h (n = 3). GAPDH was used as the control. (F–I) Bar graph showing quantitative analysis of GDF15, ACSL4, FTH1, and GPX4 (n = 3). (J–M) The values of GSH, MDA, 4-HNE, and Fe²⁺ concentrations were determined; n = 6. The error bars represent the SD. *p < 0.05 vs. control group by one-way ANOVA followed by Tukey’s post hoc analysis (*p < 0.05, **p < 0.01, and ***p < 0.001). Sham: Only laminectomy was performed.

FIGURE 2

GDF15 effectively alleviated oxidative stress-dependent neuronal ferroptosis in vitro. (A) Relative mRNA level of GDF15 after knockdown (n = 6). (B) Western blotting performed for GDF15 and ferroptosis-associated proteins including ACSL4, FTH1, and GPX4 in Hemin-activated primary neurons after transfection of KD-GDF15 or adding rGDF15 (n = 3). GAPDH was used as the control. (C–F) Bar graph showing quantitative analysis of GDF15, ACSL4, FTH1, and GPX4 (n = 3). (G) Representative immunofluorescence labeling images for GDF15 (red) and ACSL4 (green) in Hemin-activated primary neurons after transfection of KD-GDF15 or adding rGDF15 (Scale bar = 50 μm). (H–K) The values of GSH, MDA, 4-HNE, and Fe²⁺ concentrations were determined (n = 6). (L) The value of ROS was determined (n = 6). (M) Bar graph showing quantitative analysis of ROS expression (n = 6). The error bars represent the SD. **p < 0.01, ***p < 0.001, vs. control group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs. Hemin group; ^&p < 0.05, ^&⁣&&p < 0.001, vs. Hemin + KD group by one-way ANOVA followed by Tukey’s post hoc analysis.

FIGURE 3

GDF15 mitigates Hemin-induced ROS production and ferroptosis in neurons through the p62-Keap1-Nrf2 signaling pathway. (A) Relative mRNA level of p62 after knockdown (n = 6). (B) Western blotting performed for p62, Keap1, Nrf2, HO-1, ACSL4, FTH1, and GPX4 in Hemin-activated primary neurons after adding rGDF15 or transfection of sh-p62 (n = 3). β-Tubulin was used as the control. (C–I) Bar graph showing quantitative analysis of p62, Keap1, Nrf2, HO-1, ACSL4, FTH1, and GPX4 (n = 3). (J) Representative immunofluorescence labeling images for p62 (red) and Nrf2 (green) in Hemin-activated primary neurons after adding rGDF15 or transfection of sh-p62 (Scale bar = 200 μm). (K) Bar graph showing quantitative analysis of ROS expression (n = 6). (L) The value of ROS was determined (n = 6). The error bars represent the SD. **p < 0.01, ***p < 0.001, vs. control group; ^##p < 0.01, ^###p < 0.001, vs. Hemin group; ^&p < 0.05, ^&&p < 0.01, ^&⁣&&p < 0.001, vs. Hemin + rGDF15 group by one-way ANOVA followed by Tukey’s post hoc analysis.

FIGURE 4

Silencing GDF15 aggravated ferroptosis after SCI via destabilizing p62 and Nrf2 level. (A) Relative mRNA level of GDF15 in SCI mice after knockdown (n = 6). (B) Western blotting of ACSL4, FTH1, and GPX4 protein levels at 1 dpi in Sham, SCI, and SCI + KD mice (n = 3). (C–E) Bar graph showing a quantitative analysis of ACSL4, FTH1, and GPX4 (n = 3). (F) Western blotting performed for GDF15, p62, Keap1, Nrf2, and HO-1 at 1 dpi in Sham, SCI, and SCI + KD mice (n = 3). (G–K) Bar graph showing a quantitative analysis of GDF15, p62, Keap1, Nrf2, and HO-1 (n = 3). (L) Double IF of GPX4-1 (red) and NeuN (green), obtained from longitudinal sections centered around central canal at 1 dpi in Sham, SCI, and SCI + KD mice (Scale bar = 40 μm). The error bars represent the SD. **p < 0.01, ***p < 0.001, vs. Sham group; ^##p < 0.01, ^###p < 0.001, vs. SCI group by one-way ANOVA followed by Tukey’s post hoc analysis.

FIGURE 5

Inhibition of GDF15 aggravated neurological damage and neuroinflammation. (A) Neuronal death determined by TUNEL assay at 7 dpi in Sham, SCI, and SCI + KD mice (Scale bar = 200 μm). (B) Quantitative analysis of TUNEL-positive neurons. (C) Representative immunofluorescence labeling of neurofilaments for NF200 (green) and myelin sheath for MBP (red) and obtained from longitudinal sections centered around the injured core 1.5 mm at 28 dpi (Scale bar = 300 μm). (D) Quantitative analysis of NF200 positive area at 28 dpi (n = 6). (E) Quantitative analysis of MBP positive area at 28 dpi (n = 6). (F) Double immunofluorescence labeling of microglia for IBA-1 (green) and astrocytes for GFAP (red) obtained from longitudinal sections centered around the injured core 3 mm at 7 dpi (Scale bar = 300 μm). (G) Quantitative analysis of IBA-1 positive area at 7 dpi. (H) Quantitative analysis of GFAP positive area at 7 dpi. The error bars represent the SD. **p < 0.01, ***p < 0.001, vs. Sham group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs. SCI group by t-test, one-way ANOVA followed by Tukey’s post hoc analysis.

FIGURE 6

Knockdown of GDF15 interferes locomotor recovery by aggravated neuronal loss in SCI mice. (A) H&E staining images of cords centered around the injured core 3 mm obtained at 7 and 28 dpi (Scale bar = 300 μm). (B) Quantitative analysis of the defected area at 7 and 28 dpi (n = 6). (C) Representative images for Nissl staining obtained from longitudinal sections centered around the injured core 1.5 mm at 7 and 28 dpi in Sham, SCI, and SCI + KD mice (Scale bar = 200 μm). (D) Quantitative analysis of the amounts of survived neurons at 7 and 28 dpi (n = 6). (E) The BMS score post SCI in Sham, SCI, and SCI + KD mice. (F,G) Photographs of Swimming at 28 dpi, showing the worse trunk instability and uncoordinated action in SCI mice, and statistical analysis of the Louisville Swim Scale over a period of 28 days (n = 6). ***p < 0.001, vs. Sham group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, vs. SCI group by two-way ANOVA followed by Tukey’s post hoc analysis.

FIGURE 7

GDF15 alleviates SCI-induced neuronal ferroptosis by regulating the p62-Keap1-Nrf2 signaling pathway. After SCI, cracked blood vessels at the injured cords result in the lysis and destruction of erythrocytes, which release a large amount of iron. Excessive iron accumulated in neurons produces superfluous ROS and causes neuronal ferroptosis, which promotes the activation of the p62-Keap1-Nrf2 signaling pathway. Besides, GDF15 in neurons further facilitates the activation of the p62-Keap1-Nrf2 pathway by stabilizing p62 and increased Nrf2 and HO-1 inhibit the accumulation of ROS and thus alleviate neuronal ferroptosis. GDF15 is suggested as a potential target on mitigating nervous tissue loss and promoting locomotor recovery post SCI.