Chronic Deletion and Acute Knockdown of Parkin Have Differential Responses to Acetaminophen-induced Mitophagy and Liver Injury in Mice

Abstract

We previously demonstrated that pharmacological induction of autophagy protected against acetaminophen (APAP)-induced liver injury in mice by clearing damaged mitochondria. However, the mechanism for removal of mitochondria by autophagy is unknown. Parkin, an E3 ubiquitin ligase, has been shown to be required for mitophagy induction in cultured mammalian cells following mitochondrial depolarization, but its role in vivo is not clear. The purpose of this study was to investigate the role of Parkin-mediated mitophagy in protection against APAP-induced liver injury. We found that Parkin translocated to mitochondria in mouse livers after APAP treatment followed by mitochondrial protein ubiquitination and mitophagy induction. To our surprise, we found that mitophagy still occurred in Parkin knock-out (KO) mice after APAP treatment based on electron microscopy analysis and Western blot analysis for some mitochondrial proteins, and Parkin KO mice were protected against APAP-induced liver injury compared with wild type mice. Mechanistically, we found that Parkin KO mice had decreased activated c-Jun N-terminal kinase (JNK), increased induction of myeloid leukemia cell differentiation protein (Mcl-1) expression, and increased hepatocyte proliferation after APAP treatment in their livers compared with WT mice. In contrast to chronic deletion of Parkin, acute knockdown of Parkin in mouse livers using adenovirus shRNA reduced mitophagy and Mcl-1 expression but increased JNK activation after APAP administration, which exacerbated APAP-induced liver injury. Therefore, chronic deletion (KO) and acute knockdown of Parkin have differential responses to APAP-induced mitophagy and liver injury in mice.

Keywords: Acetaminophen; Autophagy; Cell Death; Liver Injury; Mitochondria; Mitophagy.

FIGURE 1.

Parkin KO mice were resistant to APAP-induced liver injury. A, WT mice were treated with 500 mg/kg APAP or saline control for 6 h, and liver cytosolic and mitochondrial fractions were isolated and analyzed by Western blot. Data from two representative mice are shown. β-Actin and voltage-dependent anion channel (VDAC) were used as loading controls for cytosolic and mitochondrial fractions, respectively. Densitometry quantification of Western blots for ubiquitin is shown. Data shown are means ± S.E. (n = 5 for each group). Results were normalized to Actin. *, p < 0.05. B, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 6 or 24 h, and blood samples were measured for serum ALT. Data shown are means ± S.E. (n ≥ 5; *, p < 0.05). C, representative H&E images are shown from WT and Parkin KO mice treated with 500 mg/kg APAP or saline control for 6 or 24 h (×100 magnification). D, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 6 and 24 h, and caspase-3 activity was measured using liver lysate. Liver lysate from a saline-treated Atg5 KO mouse was used as a positive control. Data shown are means ± S.E. (n = 3, no significant differences between groups).

FIGURE 2.

Protection in Parkin KO mice was not due to differences in APAP metabolism or oxidative stress. A, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 0.5, 1, 2, 6, or 24 h before measurement of liver GSH. Data shown are means ± S.E. (n ≥ 3; *, p < 0.05 compared with individual saline controls, no significant differences between WT and Parkin KO mice). B, WT and Parkin KO mice were treated with 500 mg/kg APAP for 6 h, and total liver lysates from individual mice were analyzed by Western blot. Gapdh was used as a loading control (HE = high exposure). C, WT and Parkin KO mice were treated as in A, and liver GSSG was measured. Data shown are means ± S.E. (n ≥ 3; *, p < 0.05 compared with WT saline control, no significant differences between WT and Parkin KO mice). D, ratio from results for GSH in A and GSSG in C are further calculated. Data shown are means ± S.E. (*, p < 0.05 compared with individual saline controls, no significant differences between WT and Parkin KO mice).

FIGURE 3.

Mitophagy occurred in both WT and Parkin KO mice after APAP treatment. A, representative EM images are shown from WT and Parkin KO mice treated with APAP (500 mg/kg) with or without CQ (60 mg/kg) for 6 h. An enlarged image of mitophagosomes is shown from the dotted line boxed area (arrows, mitophagosomes; M, mitochondria; N, nucleus). B, quantification of EM images. Data shown are means ± S.E. (n ≥ 2 mice per group, at least 10 images quantified per group; *, p < 0.05, no significant differences between WT and Parkin KO mice). C, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 6 and 24 h, and total liver lysates were analyzed for mitochondrial protein degradation by Western blot. Gapdh was used as a loading control. Results for two individual mice are shown. D and E, densitometry quantification of Western blots for Tom20 and cyclophilin D. Data shown are means ± S.E. (n = 4 for each group, no significant differences among groups). Results were normalized to Gapdh.

FIGURE 4.

Protection in Parkin KO mice may be due to decreased JNK activation. A, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 1, 2, or 6 h, and total liver lysates were analyzed by Western blot. Results from one representative mouse are shown for saline, and results from two representative mice are shown for APAP treatment. Gapdh was used as a loading control (LE, low exposure). B, densitometry quantification of pJNK. Data shown are means ± S.E. (n = 2 each for WT and Parkin KO saline, 3 each for WT and Parkin KO APAP-treated mice; *, p < 0.05 compared with WT APAP treatment at 6 h; **, p < 0.05 compared with WT APAP treatment at 24 h). Results were normalized to Gapdh. C, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 6 h, and cytosolic and mitochondrial fractions were analyzed by Western blot. CoxIV and Gapdh were used as loading controls for HM and cytosolic fractions, respectively (HE, high exposure). D, densitometry quantification of pJNK in the mitochondrial fraction. Data shown are means ± S.E. (n = 2 each for WT and Parkin KO saline treated mice, 3 each for WT and Parkin KO APAP-treated mice; *, p < 0.05 compared with WT APAP treatment at 6 h; **, p < 0.05 compared with WT APAP treatment at 24 h). Results were normalized to CoxIV.

FIGURE 5.

Parkin KO mice had increased levels of Mcl-1 proteins after APAP treatment. A, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 6 h, and total liver lysates were analyzed by Western blot. Gadph was used as a loading control. B, densitometry quantification of Western blots for Mcl-1. Data shown are means ± S.E. (n = 4 per group). Results were normalized to Gapdh. C, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 6 and 24 h. Twenty micrograms of total liver lysates were used to measure the 20 S proteasomal activity using a fluorogenic substrate. Results are presented as means ± S.E. from four different mice.

FIGURE 6.

Protection in Parkin KO mice may be due to increased hepatocyte proliferation. A, WT and Parkin KO mice were treated with 500 mg/kg APAP or saline control for 6 h, and total liver lysates were analyzed by Western blot. B, densitometry quantification of Western blots for cyclin D1. Data shown are means ± S.E. (n = 4 per group). Results were normalized to Gapdh. C, representative images from PCNA staining are shown (arrows represent PCNA-positive hepatocytes). D, PCNA staining quantification. Results are presented as % PCNA-positive hepatocytes. Data shown are means ± S.E. (n = 3, ×100 magnification, no significant differences between groups). Six images were quantified per liver tissue. E, mice were treated as in A, and total liver lysates were analyzed by Western blot. F, densitometry quantification of Western blots for PCNA. Data shown are means ± S.E. (n = 3–4 per group; *, p < 0.05). Results were normalized to β-actin.

FIGURE 7.

APAP-induced Mcl-1 degradation and necrosis in cultured mouse hepatocytes independent of Parkin. A and B, isolated hepatocytes from WT and Parkin KO mice were treated with APAP (10 mm) for 6 h in the presence or absence of Bort (50 nm). Total cell lysates were subjected to Western blot analysis. C, isolated hepatocytes from WT and Parkin KO mice were cultured for 2 and 24 h. Total cell lysates were subjected to Western blot analysis for Parkin. Representative blots from three independent experiments are shown. D, hepatocytes were first loaded with tetramethylrhodamine methyl ester (50 nm) for 15 min and then treated with APAP for 8 h. Cells were further stained with Hoechst 33342 (1 μg/ml) for 5 min followed by fluorescence microscopy. The representative overlaid images are shown. Arrows denote the cells with loss of mitochondrial membrane potential. E, number of cells with loss of mitochondrial membrane potential was quantified. Data are means ± S.E. from three independent experiments (More than 300 hundred cells were counted in each experiment from 3 to 4 different fields; *, p < 0.05.) F, WT and Parkin KO hepatocytes were treated with APAP for 24 h, and cells were stained with propidium iodide (1 μg/ml) for 5 min followed by microscopy. Representative phase-contrast images overlaid with propidium iodide signals are shown. G, percentage of PI-positive cells was quantified. Data shown are means ± S.E. from three independent experiments (at least three different images were randomly chosen from each experiment and more than 300 cells were counted; n.s., statistically no significance).

FIGURE 8.

Acute knockdown of Parkin in mouse livers impaired with APAP-induced mitophagy and exacerbated APAP-induced liver injury. A, male C57Bl/6J mice were treated either with Ad-Neg or Ad-Parkin shRNA (i.v., 1 × 10⁹ pfu per mouse) for 4 days. Total liver, pancreas, and skeletal muscle lysates were subjected to Western blot analysis. Lysate from a Parkin KO mouse liver was used a negative control. Mice were further treated with APAP (500 mg/kg) or saline for another 6 h. B, blood samples were used to measure serum ALT levels. Data shown are means ± S.E. (n = 4; *, p < 0.05). C, representative H&E images are shown (×200 magnification). Dotted line circled areas denote centrilobular necrosis. D and E, total liver lysates were subjected to Western blot analysis. Densitometry quantification of Western blots for p-JNK1 and p-JNK2 (n = 3 per group). Results were normalized to β-actin. F, representative EM images are shown. An enlarged image of a mitophagosome showing an enveloped mitochondrion and an enlarged image of an autophagosome showing enveloped cytosolic proteins are from the dotted line boxed areas (arrow = mitophagosomes, M = mitochondria, and N = nucleus). G and H, quantification of EM images for mitophagosomes and total autophagosomes. Data shown are means ± S.E. (n ≥ 20 images quantified per group; *, p < 0.05).