Loss of hepatic LRPPRC alters mitochondrial bioenergetics, regulation of permeability transition and trans-membrane ROS diffusion

Abstract

The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders.

Figure 1.

General phenotype and liver histology in normal and liver-specific LRPPRC deficient mice. Panels A and B show mean body weight (n = 30), and ratio of liver to body weight (n = 16–17) in H-Lrpprc^+/+ and H-Lrpprc^−/− mice. Panel C shows representative images of H&E staining. Loss of lobular structure and dilated vessels (top), focal necrosis and infiltration of inflammatory cells (middle), and cholestasis (bottom) are visible in the H-Lrpprc^−/− samples (arrows). Panel D shows the quantification of Oil Red’O staining intensity in individual hepatocytes from H-Lrpprc^+/+ and H-Lrpprc^−/− livers (n = 10). Panel E shows representative images of serial liver sections stained for COX (brown staining) and SDH (blue staining) activity. Panel F shows transmission electron micrographs from both mouse strains. Arrows point to large vacuolar structures and stacked cristae, which were commonly observed in H-Lrpprc^−/−mitochondria. Difference between H-Lrpprc^+/+ and H-Lrpprc^−/− was assessed with a Student t-test: **P < 0.01; ***P < 0.001.

Figure 2.

Impact of LRPPRC deficiency on the OXPHOS system. Panel A and B: SDS-PAGE blots and densitometry analysis (n = 4–6 mice per group) showing the impact of LRPPRC deficiency on SLIRP and selected components of CI (NDUFA9), CII (SDHA), CIV (COX1), and outer membrane (Porin). SDH and Actin were used as loading controls. Panel C: Activity of OXPHOS (CI, CII, CIV) and TCA cycle (CS) enzymes in isolated liver mitochondria. Enzyme activity measured spectrophotometrically (n = 4–8 mice per experimental group). Panel D: BN-PAGE blot of OXPHOS complexes in normal and H-Lrpprc^−/− mice. Antibodies to detect OXPHOS complexes were NDUFA9 (CI), SDHA (CII), UQCRC2 (CIII), COXIV (CIV) and ATP5A1 (CV). Data are representative of 4 independent experiments. Panel E: Expression of mitochondrial ribosomal subunits, and of selected mitochondrial and nuclear encoded transcripts in wild type and H-Lrpprc^−/− mice. Data were obtained at 10 weeks of age (n = 3–5). Panel F: Polyadenylated tail length analysis for COX1, COX2 and ND3 mRNA. Data shows the proportion of mRNA 3’ end displaying PolyA short, >10 and >35 chain length in wild type and Lrpprc-knockout mice. Data were obtained at 10 weeks of age (3 experimental replicates per group, using pooled RNA from 3 WT and 3 KO mice). Difference between H-Lrpprc^+/+ and H-Lrpprc^−/− was assessed using one-way ANOVA: *P < 0.05; **P < 0.01, ***P < 0.001.

Figure 3.

Impact of LRPPRC deficiency on ATP synthase (CV) activity and assembly. Panel A: Enzyme activity measured spectrophotometrically in mitochondrial extracts in absence and presence of the CV inhibitor Oligomycin (1.2 µM). Data are expressed as fold changes vs wild type values (n = 6–8). Panel B: BN-PAGE blot of DDM-solubilized mitochondria from wild type and H-Lrpprc^−/− mice. Membranes were probed with anti-ATPα Mono: CV monomers, Sub: Sub-assembled CV complexes. Panel C: Supramolecular assembly of CV revealed by in gel activity measurement. CN-PAGE was performed in duplicates for each sample. The first gel was used for in gel activity measurements (right lanes for each sample), while the other gel was stained with coomassie blue. Experiments were performed following extraction with DDM, which fully dissociates CV into monomers or with digitonin to preserve dimeric, and oligomeric CV complexes. Blots are representative of at least 3 independent experiments. Significantly different from the H-Lrpprc^+/+ group: **P < 0.01.

Figure 4.

Impact of LRPPRC deficiency on the mitochondrial bioenergetics phenotype. Panel A: Baseline state 2, state 3 (1 mM ADP), and CCCP (0.03 µM)-uncoupled respiration in liver mitochondria energized with CI (glutamate/malate [GM: 5/2.5 mM]), CII (Succinate 5 mM) substrates, or following stimulation of long-chain fatty acid oxidation (palmitoyl-CoA [20μM]) (n = 5–9). Panel B: Maximal state 3 respiration in isolated liver mitochondria energized with CIV substrates (ascorbate/TMPD: 9mM/0.9mM) (n = 5). Panel C: Titration of CIV-driven (Ascorbate/TMPD 9/0.9 mM) state 3 respiration with potassium cyanide (KCN) in wild type and H-Lrpprc^−/− mitochondria. Best fit and 95% confidence intervals are shown for each dataset (n = 5).

Figure 5.

Impact of LRPPRC deficiency on the permeability transition pore: Panel A-C: Representative tracings of mitochondrial calcium retention capacity (CRC) in isolated liver mitochondria exposed to consecutive pulses of calcium (2.5 μmole/mg protein per pulse). All experiments were performed in the presence of Succinate (5 mM), Rotenone (1 µM) and Pi (10 mM). In panel B and C, the incubation buffer was respectively supplemented with Cyclosporin-A (1µM), or a combination of ADP (12 µM), MgCl₂ (0.6 mM) and Oligomycin (27 µM). Panel D: Average Calcium Retention Capacity observed in the three experimental conditions described in A-C (n = 8–11). Panel E: Immunoblot and densitometric analysis of Cyclophilin-D (CypD) content in isolated liver mitochondria from wild type and H-Lrpprc^−/− mitochondria. Mitochondrial lysates from wild-type and CypD deficient Ppif^−/− mice were run as controls control (n = 6). Panel F: Immunoblot and densitometric analysis of OSCP content in whole mitochondrial lysates and OXPHOS extracts obtained following digitonin treatment. Significantly different from wild type mice: **P < 0.01, ***P < 0.001; Significantly different from control condition: Φ: P < 0.001.

Figure 6.

Impact of LRPPRC deficiency on mitochondrial H₂O₂ dynamics: Panel A: Net H₂O₂ release from mitochondria under state 2 and state 3 (1 mM ADP) conditions in the presence of substrates for CI + CII (G/M/Succ: 5/2.5/5 mM) (n = 8–16). In some experiments (n = 3), net H₂O₂ release was measured in the presence of succinate alone (Succ: 5 mM) and rotenone (1 µM) was added to confirm that H₂O₂ release occurred mainly through reverse electron backflow to CI. Panel B: Mitochondrial membrane potential measured on respiring isolated liver mitochondria energized with complex I + II (G/M/Succ: 5/2.5/5 mM) or complex II (succinate 5mM) substrates. (n = 6). Panel C: Immunoblot and densitometric analysis of SOD2 and catalase content in mitochondria from wild type and H-Lrpprc^−/− mice (n = 6). Panel D: Kinetics of scavenging of an exogenous H₂O₂ load (3 nmoles) by mitochondria energized with CI substrates (Gutamate/Malate: 50/20 µM) (n = 6). Panel E: Net H₂O₂ release from mitochondria under state 2 conditions in the presence of substrates for CI + CII (G/M/Succ: 5/2.5/5 mM). Following baseline measurements, digitonin was progressively added and changes in H₂O₂ release were monitored (n = 6). Insets in panel E shows progressive inhibition of respiration in H-Lrpprc^+/+(black tracing) H-Lrpprc^−/− (grey tracing) in response to progressive permeabilization with digitonin. Panel F: H₂O₂ production measured in sub-mitochondrial particles (SMP) in the presence of succinate (5 mM) alone, or with Antimycin-A (AA: 2 µM) (n = 4). Panel G: Immunoblot and densitometric analysis of AQP8 content in mitochondria from wild type and H-Lrpprc^−/− mitochondria (n = 6). Significantly different from wild type mice: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7.

Impact of LRPPRC deficiency on mitochondrial membrane lipid composition: Panel A: Volcano plot showing entities that were identified as differentially expressed, either up or downregulated, in H-Lrpprc^−/− mitochondria according to the following selection criteria: fold change > 2 and corrected p value < 0.05. Panel B: Individual lipid species found to be significantly up or down-regulated on H-Lrpprc^−/− mitochondria. Each dot represents a Log2-transformed ratio of an individual H-Lprrpc^−/− mitochondria relative to the mean value obtained in the wild type group (n = 6 per group).