Double deletion of PINK1 and Parkin impairs hepatic mitophagy and exacerbates acetaminophen-induced liver injury in mice

Abstract

Mitochondria damage plays a critical role in acetaminophen (APAP)-induced necrosis and liver injury. Cells can adapt and protect themselves by removing damaged mitochondria via mitophagy. PINK1-Parkin pathway is one of the major pathways that regulate mitophagy but its role in APAP-induced liver injury is still elusive. We investigated the role of PINK1-Parkin pathway in hepatocyte mitophagy in APAP-induced liver injury in mice. Wild-type (WT), PINK1 knockout (KO), Parkin KO, and PINK1 and Parkin double KO (DKO) mice were treated with APAP for different time points. Liver injury was determined by measuring serum alanine aminotransferase (ALT) activity, H&E staining as well as TUNEL staining of liver tissues. Tandem fluorescent-tagged inner mitochondrial membrane protein Cox8 (Cox8-GFP-mCherry) can be used to monitor mitophagy based on different pH stability of GFP and mCherry fluorescent proteins. We overexpressed Cox8-GFP-mCherry in mouse livers via tail vein injection of an adenovirus Cox8-GFP-mCherry. Mitophagy was assessed by confocal microscopy for Cox8-GFP-mCherry puncta, electron microscopy (EM) analysis for mitophagosomes and western blot analysis for mitochondrial proteins. Parkin KO and PINK1 KO mice improved the survival after treatment with APAP although the serum levels of ALT were not significantly different among PINK1 KO, Parkin KO and WT mice. We only found mild defects of mitophagy in PINK1 KO or Parkin KO mice after APAP, and improved survival in PINK1 KO and Parkin KO mice could be due to other functions of PINK1 and Parkin independent of mitophagy. In contrast, APAP-induced mitophagy was significantly impaired in PINK1-Parkin DKO mice. PINK1-Parkin DKO mice had further elevated serum levels of ALT and increased mortality after APAP administration. In conclusion, our results demonstrated that PINK1-Parkin signaling pathway plays a critical role in APAP-induced mitophagy and liver injury.

Role and Mechanisms of Mitophagy in Liver Diseases

“…These results imply the protective role of autophagy/mitophagy in attenuating the hepatotoxicity induced by an APAP overdose. Interestingly, we found that Parkin knockout mice are resistant to APAP-induced liver injury, while PINK1/Parkin double-knockout mice develop more severe liver injury with markedly decreased mitophagy after APAP administration compared with wild type mice [155,159]. This is likely because PINK1-mediated mitophagy still occurs in the absence of Parkin, which may compensate for the lack of Parkin.…”

“…However, acute knockdown of Parkin accelerated APAP-induced liver injury in mice, inferring that under the acute knockdown of the Parkin time window the mice do not have sufficient time to adapt to the acute loss of Parkin [157]. Indeed, our recent work revealed that very low levels of mitophagy were detected in the PINK1/Parkin double-knockout mouse livers with or without APAP treatment, and the PINK1/Parkin double-knockout mice have the most severe liver injury compared with wild-type or either of the single knockout mice [159]. It should be noted that although PINK1/Parkin-mediated mitophagy serves as a protective mechanism against APAP-induced hepatotoxicity, other potential Parkin-independent pathways may still need to be further identified.…”

“…It should be noted that most outer mitochondrial membrane proteins are degraded by the ubiquitin proteasome system during mitophagy, whereas the degradation of inner mitochondrial proteins are more specific for mitophagy [12]. Nonetheless, Cox8-EGFP-mCherry, which targets the inner mitochondrial membrane, may be more specific than the Mito-QC that has been developed for monitoring mitophagy [159,224]. Although the pH-dependent fluorescent marker has great value for the monitoring and quantifying of mitophagy, it also has the limitations of many other imaging-based assays such as the lack of data robustness and consistency among different investigators scoring the mitophagy events.…”

“…ALD [114,116,119,122] NAFLD [134,140] DILI [155,157,159] HBV/HCV [192] Cancer [216] The expression level of fluorescent proteins varies in different cells/tissues Not robust and easy to be dependent on individuals who are performing the quantification. Also, the half-life of the red puncta in the lysosomes may be dependent on cellular context and conditions, e.g., the activities of the lysosomal proteases NAFLD [86] DILI [159] Cancer [216] 6. Summary and Future Perspectives…”

Toxicological Property of Acetaminophen: The Dark Side of a Safe Antipyretic/Analgesic Drug?

“…108,111,112) Moreover, recent reports on riddance of damaged mitochondria by mitophagy have confirmed the pathophysiological importance of mitochondria. 113) Ni et al 114) demonstrated protection from APAP hepatotoxicity by removing APAP protein adducts by autophagy in mouse model. Wang et al 115) also reported that double deletion of PINK1 and Parking impaired mitophagy pathway and hence exacerbated APAP-induced liver injury and mortality in mice.…”