Cardiolipin-Dependent Mitophagy Guides Outcome after Traumatic Brain Injury

Abstract

Mitochondrial energy production is essential for normal brain function. Traumatic brain injury (TBI) increases brain energy demands, results in the activation of mitochondrial respiration, associated with enhanced generation of reactive oxygen species. This chain of events triggers neuronal apoptosis via oxidation of a mitochondria-specific phospholipid, cardiolipin (CL). One pathway through which cells can avoid apoptosis is via elimination of damaged mitochondria by mitophagy. Previously, we showed that externalization of CL to the mitochondrial surface acts as an elimination signal in cells. Whether CL-mediated mitophagy occurs in vivo or its significance in the disease processes are not known. In this study, we showed that TBI leads to increased mitophagy in the human brain, which was also detected using TBI models in male rats. Knockdown of CL synthase, responsible for de novo synthesis of CL, or phospholipid scramblase-3, responsible for CL translocation to the outer mitochondrial membrane, significantly decreased TBI-induced mitophagy. Inhibition of mitochondrial clearance by 3-methyladenine, mdivi-1, or phospholipid scramblase-3 knockdown after TBI led to a worse outcome, suggesting that mitophagy is beneficial. Together, our findings indicate that TBI-induced mitophagy is an endogenous neuroprotective process that is directed by CL, which marks damaged mitochondria for elimination, thereby limiting neuronal death and behavioral deficits.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) increases energy demands leading to activation of mitochondrial respiration associated with enhanced generation of reactive oxygen species and resultant damage to mitochondria. We demonstrate that the complete elimination of irreparably damaged organelles via mitophagy is activated as an early response to TBI. This response includes translocation of mitochondria phospholipid cardiolipin from the inner membrane to the outer membrane where externalized cardiolipin mediates targeted protein light chain 3-mediated autophagy of damaged mitochondria. Our data on targeting phospholipid scramblase and cardiolipin synthase in genetically manipulated cells and animals strongly support the essential role of cardiolipin externalization mechanisms in the endogenous reparative plasticity of injured brain cells. Furthermore, successful execution and completion of mitophagy is beneficial in the context of preservation of cognitive functions after TBI.

Keywords: apoptosis; autophagy; cardiolipin; mitophagy; neuroprotection; phospholipid.

Figure 1.

Mitophagy induction after traumatic brain injury. A, B, Representative TEM images of neurons from human brains. Mitochondria within autophagosome-like structures, such as a double membrane (arrow), were observed in TBI brain tissue. A significant difference in the number of autophagic vacuoles containing mitochondria per neuron (obtained from TEM images) was observed between TBI (n = 5) and Ctrl (Ctrl n = 3, TBI n = 5; mean ± SD; t = 6.723; *p < 0.05 vs Ctrl by t test). C, The ratio of mtDNA/gDNA was assessed by real-time PCR, reflecting the relative number of mitochondria in human samples (Ctrl n = 3, TBI n = 5, mean ± SD; t = 8.534; *p < 0.05 vs Ctrl by t test). D, E, Western blot of autophagic and mitochondrial markers obtained from TBI (n = 5) and control (n = 3) patients. GAPDH was used as the loading control. Bar graphs show the results of analysis (by band-density analysis) of p62, LC3-II, TOM40, MnSOD, and COXIV (Ctrl n = 3, TBI n = 5, mean ± SD; p62 t = 3.325, LC3-II t = 7.364, TOM40 t = 4.127, MnSOD t = 2.633, COXIV t = 3.172; *p < 0.05 vs Ctrl by t test). F, G, TEM images of neurons in the rat brains 1 h after CCI; note that mitochondria were engulfed by autophagic vacuoles. Scale bar, 0.1 μm. Autophagic vacuoles containing damaged mitochondria were quantified after CCI (Ctrl n = 3, TBI n = 5; mean ± SD; t = 4.571; *p < 0.05 vs Ctrl by t test). H, I, Detection of mitophagy in the injured cortex of the rat after CCI (in vivo TBI model). CCI increased colocalization of GFP-LC3 and mitochondria (arrows) versus control (n = 5; mean ± SD; F_(4,20) = 20.317; *p < 0.05 vs Ctrl by one-way ANOVA and Tukey's post hoc test). Scale bar, 10 μm. J–L, CCI-induced decrease of mitochondrial markers (TOM40, MnSOD, and COXIV) was partially reversed by Baf-A1-mediated inhibition of autophagosomal-lysosomal degradation; GAPDH was set as the standard (n = 3; mean ± SD; p62: F_(4,8) = 9.532,TOM40: F_(4,8) = 7.332; *p < 0.05 vs control; #p < 0.05 vs CCI by one-way ANOVA and Tukey's post hoc test). Baf-A1, Bafilomycin A1; mt, mitochondria; TBI, traumatic brain injury; CCI, controlled cortical impact; TBI-AVs, autophagic vacuoles; h, hour; TEM, Transmission electron microscopy; Ctrl, control group.

Figure 2.

TBI-induced mitophagy in vitro. A, B, Trauma increased the colocalization of GFP-LC3 and mitochondria (arrows) in primary cortical neurons subjected to stretch injury (in vitro TBI model: n = 5; mean ± SD; t = 6.712; *p < 0.05 vs Ctrl by t test). Scale bar, 10 μm. C, D, Representative electron-micrographs of primary cortical neurons. The arrow indicates mitochondria engulfed by vacuolar structures (n = 3; mean ± SD; t = 3.425; *p < 0.05 vs control by t test). Scale bar, 0.2 μm. E–H, Western blot of general autophagy markers and mitochondrial proteins in primary cortical neurons subjected to stretch injury. β-Actin was used as the loading control [(E) n = 3; mean ± SD; F_(4,10) = 4.362; *p < 0.05 vs control by one-way ANOVA and Tukey's post hoc test); (G) n = 3; mean ± SD; F_(4,10) = 12.143; *p < 0.05 vs control by one-way ANOVA and Tukey's post hoc test; (H) n = 3; mean ± SD; F_(4,10) = 7.336; *p < 0.05 vs control by one-way ANOVA and Tukey's post hoc test].

Figure 3.

Temporal course of mitophagy and apoptosis after TBI. A–C, Both general autophagy assessed by LC3-II expression and mitophagy assessed by loss of OMM (TOM 40), IMM (COX IV), and matrix (MnSOD) proteins were induced in the cortex as early as 1 h after CCI and persisted for 24 h after injury [(B) n = 5; mean ± SD; F_(4,20) = 16.334; *p < 0.05 vs Ctrl by one-way ANOVA and Tukey's post hoc test; (C) mean ± SD; F_(4,20) = 7.617; *p < 0.05 vs Ctrl by one-way ANOVA and Tukey's post hoc test]. D–F, Apoptosis assessed by TUNEL staining (n = 5) and release of cyt c from mitochondria to cytosol in contusional cortex did not occur until 6 h after CCI (n = 5; mean ± SD; F_(4,20) = 23.143; *p < 0.05 vs Ctrl by one-way ANOVA and Tukey's post hoc test). Scale bar, 100 μm. CYTO, Cytosolic fraction; MITO, mitochondrial fraction.

Figure 4.

Mitophagy in the hippocampus after TBI. A, Representative electron micrographs of injured and sham neurons at 1 h after injury. A mitophagosome containing partially degraded mitochondria is indicated by the arrow. Scale bar, 0.1 μm. B, Mitophagosome number was increased after CCI compared with control (n = 3; mean ± SD; t = 13.761; *p < 0.05 vs control by t test). C–E, The levels of autophagic and mitochondrial markers were assessed by Western blot after TBI [(D)n = 3; mean ± SEM; F_(4,10) = 5.417; *p < 0.05 vs control by one-way ANOVA and Tukey's post hoc test; (E) n = 3; mean ± SEM; F_(4,10) = 13.672; *p < 0.05 vs control by one-way ANOVA and Tukey's post hoc test].

Figure 5.

PLS3 knockdown inhibits TBI-induced mitophagy. A, Exposure of CL on mitochondrial surface and phosphorylation of PLS3 (B) are increased in human brains after TBI. (Ctrl n = 3 and TBI n = 5; mean ± SD; t = 2.534; p < 0.05 vs Ctrl by t test). C, PLS3 knockdown significantly attenuated CCI-induced CL externalization (n = 5; mean ± SD; F_(2,12) = 10.530; *p < 0.05 vs control and #p < 0.05 vs control by one-way ANOVA and Tukey's post hoc test). D–F, CCI elicited increases in LC3-II levels and decreases in mitochondrial protein content, which was reversed by siRNA knockdown of PLS3. Note that PLS3 knockdown had no effect on the expression of the general autophagy marker LC3-II [n = 5; (E) mean ± SEM; F_(4,20) = 3.513; *p < 0.05 vs Ctrl and p > 0.05 CCI+siRNA PLS3 vs CCI+siRNA Ctrl by one-way ANOVA and Tukey's post hoc test; (F) mean ± SEM; F_(4,20) = 13.835; *p < 0.05 vs Ctrl and #p < 0.05 vs CCI+siRNA Ctrl by one-way ANOVA and Tukey's post hoc test]. G, Traumatic stimuli activated PLS3 phosphorylation and (H) increased surface exposure of CL at 3 h after injury in stretch model (n = 5; mean ± SEM; F_(2,12) = 23.153; *p < 0.05 vs Ctrl and #p < 0.05 vs CCI+siRNA Ctrl by one-way ANOVA and Tukey's post hoc test). PT, Phosphothreonine; IP, immunoprecipitation; IB, immunoblotting; PLS3, phospholipid scramblase-3.

Figure 6.

Analysis of CL-mediated mitophagy in response to traumatic injury. A–C, TBI-induced decrease of mitochondrial proteins was reversed by siRNA targeting CLS in in vitro experiment [(B) n = 5; mean ± SD; F_(4,20) = 13.732; *p < 0.05 vs 1 h with siRNA CLS and #p < 0.05 vs 0.5 h with siRNA CLS by one-way ANOVA and Tukey's post hoc test; (C) n = 5; mean ± SEM; F_(4,20) = 9.538; *p < 0.05 vs 1 h with siRNA CLS and #p < 0.05 vs 0.5 h with siRNA CLS by one-way ANOVA and Tukey's post hoc test]. D–F, Knockdown of CLS attenuated mitophagy after CCI. Both general autophagy assessed by LC3-II expression and mitophagy assessed by loss of OMM (TOM 40) and IMM (COX IV) proteins were induced in cortex at 1 and 3 h after injury [n = 3; (E) mean ± SEM; F_(4,10) = 10.679; *p < 0.05 vs Ctrl and p > 0.05 CCI+siRNA PLS3 vs CCI+siRNA Ctrl by one-way ANOVA and Tukey's post hoc test; (F) mean ± SEM; F_(4,10) = 15.139; *p < 0.05 vs Ctrl and #p < 0.05 vs CCI+siRNA Ctrl by one-way ANOVA and Tukey's post hoc test]. G, H, Mitophagy was assessed by colocalization of GFP-LC3 with mitochondrial protein COX-IV (n = 5) in GFP-LC3 transgenic rats transfected for 72 h with CLS siRNA or control siRNA intracerebroventricularly. Neuronal nuclei were stained with anti-NeuN antibody in blue (mean ± SD; F_(4,20) = 25.460; *p < 0.05 vs control by t test; #p < 0.05 vs CCI + siRNA Ctrl by one-way ANOVA and Tukey's post hoc test). Scale bar, 10 μm. CLS, cardiolipin synthase.

Figure 7.

Suppression of mitophagy worsens TBI-induced neuronal injury and behavioral deficits. A, B, Mdivi-1 did not affect general autophagosome biogenesis induced by CCI at 1 h post-injury (n = 3; mean ± SD; F_(3,8) = 22.742; *p < 0.05 vs control and p > 0.05 CCI vs CCI+Mdivi-1 by one-way ANOVA and Tukey's post hoc test). A, C, The loss of mitochondrial proteins induced by CCI was partly reversed by mdivi-1. GAPDH was used as the loading control (n = 3; mean ± SD; F_(3,8) = 12.583; *p < 0.05 vs control and #p < 0.05 vs CCI by one-way ANOVA and Tukey's post hoc test). D, E, Apoptotic cell death was assessed by TUNEL staining from five random fields in the injured cortex (n = 5; mean ± SD; F_(5,24) = 40.362; ^#p < 0.05 vs CCI, 1 h by t test). Scale bar, 100 μm. F, 3-MA or mdivi-1 increased the level of cleaved-caspase-3 after CCI. G, Analysis of cortical lesion volume 7 d after TBI in Sprague-Dawley rats (n = 5; mean ± SD; F_(2,15) = 4.163; *p < 0.05 vs CCI+V by one-way ANOVA and Tukey's post hoc test). H, Time spent (seconds) on the balance beam apparatus before and after CCI or sham (n = 8–12; mean ± SD; F_(5,300) = 54.362; *p < 0.05 vs CCI + Vehicle by one-way ANOVA and Tukey's post hoc test). I, Latency (seconds) till rats locate a hidden (submerged) and visible platform on post-TBI Days 11–15 (n = 8–12; mean ± SD; F_(5,300) = 72.128; *p < 0.05 vs CCI+Vehicle by one-way ANOVA and Tukey's post hoc test). J, NOR task performance 19 d after sham or CCI injury (n = 8–12 per group; mean ± SEM; F_(5,36) = 9.714; *p < 0.05 vs CCI+Vehicle by one-way ANOVA and Tukey's post hoc test). V, Vehicle.

Figure 8.

PLS3 knockdown exacerbates neuronal injury and behavioral deficits after TBI. A, Assessment of neurodegeneration by FJC staining. Neurodegeneration observed in the injured cortex 1 week after CCI was exacerbated by PLS3 downregulation. Scale bar, 100 μm. B, Quantification of FJC staining showed significantly higher neurodegeneration in the CCI + siRNA PLS3 group (n = 5; mean ± SD; F_(3,16) = 32.461; *p < 0.05 vs CCI + siRNA Ctrl by one-way ANOVA and Tukey's post hoc test). Rats were transfected with control or PLS3 siRNA 72 h before CCI and measurements were obtained 24 h after injury. Insert: The efficacy of PLS3 knockdown evaluated by Western blot, n = 5/group. C, Caspase-3 activity of pericontusional cortex (n = 5; means ± SD; F_(3,16) = 17.227 *p < 0.05 vs CCI + siRNA Ctrl by one-way ANOVA and Tukey's post hoc test). D, Analysis of lesion volume (both cortical and hippocampal areas using H&E staining) 7 d after CCI (n = 5; mean ± SD; F_(3,16) = 10.325; *p < 0.05 vs CCI + V by one-way ANOVA and Tukey's post hoc test). Scale bar, 500 μm. E, Time spent (in seconds) on the balance beam apparatus before and after CCI or sham injury (n = 8–12; mean ± SD; F_(3,200) = 72.671; *p < 0.05 vs CCI + siRNA Ctrl by one-way ANOVA and Tukey's post hoc test). F, Latency (in seconds) till rats locate a hidden (submerged) platform on post-TBI Days 11–15 (n = 8–12; F_(3,200) = 14.271; *p < 0.05 vs CCI+ siRNA Ctrl by one-way ANOVA and Tukey's post hoc test). G, NOR task performance 19 d after sham or CCI injury (n = 8–12; mean ± SEM; F_(3,36) = 45.461; *p < 0.05 vs CCI 9+ siRNA Ctrl by one-way ANOVA and Tukey's post hoc test). H, Schematic diagram summarizing the proposed role of CL in mitophagy after TBI. FJC, Fluoro jade-C.