| dc.description.abstract | Acetaminophen (APAP) is the leading cause of acute liver failure in the United States, and alcoholic liver disease (ALD) is a worldwide health problem that claims two million lives per year. Currently, the only cure for either disease is liver transplantation in severe disease states. Therefore, new therapeutic options for treatment of these liver diseases are greatly needed. To develop new therapeutic options, the mechanisms involved in APAP and alcohol-induced liver toxicities must be better understood. We previously demonstrated that autophagy was protective against both APAP and alcohol-induced liver injuries by removing damaged mitochondria by mitophagy, which is a selective form of autophagy specific for mitochondria. However, the mechanisms for induction of mitophagy in the liver are unknown. Parkin is an E3 ubiquitin ligase that is well known to be required for mitophagy induction in mammalian cell models after mitochondrial depolarization. Therefore, we evaluated the role of Parkin in inducing mitophagy as a protective mechanism against APAP and alcohol-induced liver injuries. For alcohol treatment, acute-binge and chronic-plus-binge (Gao-binge) models were used. First, we found that APAP and alcohol produced opposite responses in Parkin KO mice. Parkin KO mice were protected after APAP treatment, but they had more liver injury and steatosis after alcohol treatment compared to WT mice. It is well known that c-Jun N-terminal kinase (JNK) activation exacerbates APAP-induced liver injury, and it has recently been shown that myeloid leukemia cell differentiation protein (Mcl-1) mediates protection against APAP-induced liver injury. In addition, liver regeneration has been shown to be the most important repair mechanism for APAP-induced liver injury. We found that Parkin KO mice had decreased JNK activation, increased Mcl-1 expression, and increased hepatocyte proliferation after APAP treatment in their livers compared to WT mice. In contrast to protection after APAP treatment, Parkin KO mice were more susceptible to alcohol-induced liver injury than WT mice because of Parkin’s role in maintaining a healthy population of mitochondria, likely through activation of mitophagy. Alcohol caused greater mitochondrial damage in Parkin KO livers compared to WT livers. Parkin KO mice had severely swollen and damaged mitochondria that lacked cristae after alcohol treatment, which were not seen in WT mice. In addition, Parkin KO mice had decreased mitophagy, β-oxidation, mitochondrial respiration, and cytochrome c oxidase activity after acute-binge alcohol treatment compared to WT mice. Furthermore, Parkin KO mouse liver mitochondria had less capacity to adapt to Gao-binge treatment compared to WT mouse liver mitochondria. The fact that Parkin KO mice were protected against APAP-induced liver injury but had increased liver injury and steatosis after alcohol treatment compared to WT mice suggests that Parkin has multiple roles in maintaining cellular homeostasis in addition to its role in initiation of mitophagy, and these various roles my differ in importance depending on the amount and type of liver injury produced. Therefore, Parkin’s roles in regulating proliferation, JNK activation, and Mcl-1 expression were likely more important than mitophagy during APAP-induced liver injury while its role in mitophagy induction to maintain mitochondrial homeostasis was likely more important during alcohol-induced liver injury. Second, we surprisingly found that even though Parkin has been shown to be required for mitophagy induction in in vitro models, Parkin was not essential for mitophagy induction in the liver. We found that mitophagy still occurred in Parkin KO mice based on electron microscopy analysis after APAP and alcohol treatments. However, mitophagy levels were reduced in Parkin KO mice compared to WT mice, and Parkin translocated to mitochondria in WT mouse livers after both APAP and alcohol treatments. These results suggest that Parkin-induced mitophagy is still likely an important protective mechanism in the liver even though compensatory adaptive mechanisms exist for mitophagy induction in the absence of Parkin. In addition, these compensatory mechanisms may not be as efficient as Parkin in inducing mitophagy in the liver because mitophagy levels were reduced in Parkin KO mice compared to WT mice after APAP and alcohol treatments. Parkin-independent mechanisms for mitophagy induction in the liver are currently unknown, but they may involve activation of other proteins known to mediate mitophagy, such as Mul1, which has been shown to act in parallel to the Parkin pathway in Drosophila. Third, we found that whole-body knockout of Parkin and acute knockdown of Parkin had opposite responses to APAP overdose. While Parkin KO mice were protected against APAP-induced liver injury compared to WT mice, acute knockdown of Parkin in mouse livers resulted in increased liver injury compared to WT mice after APAP treatment. Whole-body Parkin KO mice were protected because they had increased Mcl-1 and proliferation levels and decreased JNK activation, and mice with acute knockdown had exacerbated APAP-induced liver injury because they had reduced Mcl-1 and proliferation levels and increased JNK activation. In addition, mitophagy was reduced in both Parkin KO mice and mice with acute knockdown of Parkin compared to WT mice. However, mitophagy levels were only slightly reduced in whole-body knockout mice while they were significantly reduced in mice with acute Parkin knockdown. These opposite responses between Parkin whole-body KO and acute knockdown mice were likely due to a lack of time to develop compensatory and adaptive mechanisms in the acute knockdown mice that were present in the whole-body knockout mice. Overall, our findings indicate that Parkin-induced mitophagy is likely a mechanism of protection in the liver, but compensatory mechanisms exist for induction of mitophagy in the absence of Parkin. However, these compensatory mechanisms may only play a minor role in the liver. In addition, Parkin has multiple roles in maintaining cellular homeostasis in addition to mitophagy, which causes Parkin KO mice to respond differently to various types of liver injury. However, our findings from Parkin KO mice likely better reflect physiological conditions in people that have chronic loss of Parkin function, such as Autosomal-Recessive Parkinson’s disease patients, who have mutations in the Park2 (Parkin) gene. Finally, our results indicate that evaluation of drug-induced liver injury mechanisms using whole-body knockout mice should be interpreted with caution due to adaptive and compensatory mechanisms that may be activated in knockout mice. In addition, modulating Parkin-mediated mitophagy may be a promising therapeutic approach for targeting drug and alcohol-induced liver injuries. | |
