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.
Diabetic peripheral neuropathy (DPN) is a common complication of diabetes that affects about 70% of diabetic patients, leading to infection, inflammation, amputation, and even death. The ...pathophysiology of DPN is tightly linked to a decrease in mitochondrial bioenergetics (mtBE) in sensory neurons. Neuronal mitochondrial dysfunction impairs numerous cellular functions which requires a clearance mechanism, such as mitophagy, to remove damaged mitochondria and maintain homeostasis. Mitophagy is a recently emerging novel therapeutic target in neuronal disorders as well as diabetic complications. Studies in animal models of DPN have found an accumulation of fragmented mitochondria, which may indicate an impairment in mitochondrial quality control. Two proteins involved in mitochondrial quality control and DPN are heat shock protein 70 (Hsp70) and thioredoxin-interacting protein (TXNIP). Previously reported in our lab, in vivo treatment with the heat shock protein (Hsp) 90 modulator, KU-596, can reverse symptoms of DPN, which correlates with improving mtBE in diabetic sensory neurons in an Hsp70-dependent manner. Additionally, our lab has shown elevated Txnip expression in a DPN mouse model. Although the interaction of Txnip and Hsp70 in the mitophagy pathway has not been fully investigated, we know that both are multifunctional proteins involved in several interweaving pathways. Thus, the hypothesis of these studies is that the interaction of Hsp70 with Txnip can improve mtBE and DPN by regulating neuronal mitophagy. Furthermore, the aim of this study was to also investigate whether mitophagy plays an important role in the efficacy of KU-596 in improving DPN. To investigate if mitophagy is a mechanism by which KU-596 to improve indices of DPN and for studying the relationship of Hsp70, Txnip, and mitophagy in a diabetic model, we utilized the Mito-QC (MQC) mouse model and generated 3 mouse lines- MQC, MQC x Hsp70 KO, and MQC x Txnip KO. The MQC mice express a GFP-mCherry fusion protein localized on the outer mitochondrial membrane that enables the visualization of mitophagy. As damaged mitochondria are internalized into the phagolysosomes, the acidic environment of the organelle quenches the fluorescence signals from the GFP leading to the visualization of red puncta from the mCherry signaling that can be visualized and quantified as a measure of the extent of mitophagy. Diabetic MQC, MQC x Hsp KO, and MQC x Txnip KO mice each demonstrated an increase in mitophagy levels. To determine the effect of KU-596 treatment, diabetic mice of all 3 mice lines were treated with a daily dose of 1 mg/kg KU-596 for 16 weeks. Treatment with KU-596 decreased mitophagy levels in only the MQC mouse line, which correlates with the mtBE data, showing improvements of mtBE in an Hsp70- and Txnip-dependent manner. Thus, KU-596 requires Hsp70 to slow the progression of DPN through sensory and mitochondrial function but does not necessarily require Txnip to improve sensory function.
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