Mitochondria damage takes on a critical part in acetaminophen (APAP)-induced necrosis

Mitochondria damage takes on a critical part in acetaminophen (APAP)-induced necrosis and liver organ injury. Red1 KO, Parkin KO and WT mice. We just discovered gentle 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 PU-H71 cell signaling 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. and cultured mammalian cell models suggest a linear PINK1-Parkin mitophagy pathway, which places PINK1 upstream of Parkin [15,20]. However, recent evidence suggests a new model that PINK1 alone can also induce mitophagy impartial of Parkin via directly recruit NDP52 and OPTN, two other mitophagy receptor proteins, to mitochondria [21]. Although we now understand extensively the molecular details by which PINK1-Parkin regulates mitophagy, PU-H71 cell signaling most of the known mechanisms are derived from cell culture studies that overexpress exogenous Parkin. Due to the lack of reliable quantitative mitophagy assays, relatively few studies were conducted to determine the role of PINK1-Parkin in mitophagy under pathophysiologically relevant conditions. We recently exhibited that APAP increases Parkin translocation to mitochondria, which is usually associated with increased ubiquitination of mitochondrial proteins and mitophagy in mouse livers [8]. These data imply that Parkin-mediated mitophagy may be protective against APAP-induced liver injury by removing damaged mitochondria. Unexpectedly, we also found that mitophagy still occurs in APAP-treated Parkin knockout (KO) mouse livers and that Parkin KO mice are resistant to APAP-induced liver injury [11], suggesting other compensatory mechanisms may be activated to induce mitophagy in PU-H71 cell signaling Parkin KO mouse livers. The aim of the present study was to determine the role of PINK1 and Parkin in APAP-induced mitophagy and liver injury, and whether PINK1-mediated mitophagy would serve as a compensatory mechanism in the absence of Parkin in APAP-treated mouse livers. To achieve a more reliable quantitative measure of mitophagy in mouse livers, we generated an adenovirus vector that carries a mitochondrial inner membrane-targeted tandem GFP-mCherry fusion protein. To determine the possible reciprocal compensatory role of PINK1 and Parkin in APAP-induced mitophagy and liver injury, we also generated PINK1 and Parkin double KO (DKO) mice. We found that APAP-induced mitophagy was significantly blunted in the PINK1 and Parkin DKO mice. As a result, PINK1 and Parkin DKO mice experienced more severe liver damage and increased mortality compared with either IL6 antibody wild-type (WT) mice or single PINK1 KO or Parkin KO mice after APAP. 2.?Materials and methods 2.1. Antibodies and reagents The following antibodies were utilized for western blot analysis: Parkin (Santa-Cruz, SC-32282), Ubiquitin (Santa Cruz, SC-8017), p62 (Abnova, H00008878-M01), -Actin (Sigma, A5441), Cyp2e1 (Abcam, ab19140), phosphorylated JNK (4668S), JNK (BD, 554285), Oxphos rodent antibody cocktail (Abcam, ab110413), and voltage-dependent anion channel (VDAC) (Calbiochem, 529534). The APAP-adduct antibody was a gift from Dr. Lance Pohl (NIH) [22]. Horseradish peroxidase-conjugated antibodies were from Jackson ImmunoResearch Lab. Adenovirus (Ad) Cox8-GFP-mCherry was produced in collaboration with Vector Biolabs (Malvern, PA). In situ cell death detection kit (Cat# 11684809910) was purchased from Roche. The kit for alanine aminotransferase (ALT) assay was purchased from Pointe Scientific (A7526-450). APAP and other chemicals were either purchased from Sigma-Aldrich or Thermo Fisher Scientific. 2.2. Animal experiments WT C57BL/6J, PINK1 KO (stock# 017946) and Parkin KO (Stock# 006582) were purchased from your Jackson Laboratory. PINK1 and Parkin DKO mice were generated by crossing PINK1 KO mice with Parkin KO mice. Atg5 Flox/Flox (Atg5 F/F) mice (C57BL/6/129) were generated by Dr. N. Mizushima and were backcrossed with C57BL/6J for another 10 generations before further crossing them with Albumin-Cre mice (Alb-Cre, C57BL/6) (Jackson Laboratory) as explained previously [23]. Man 8 to 12 week-old mice had been treated with APAP (500?mg/kg) or saline by intraperitoneal shot. Afterwards, mice had been euthanized at 2, 6,.