Jessica A. Williams

Mitochondrial dynamics and mitochondrial quality control.

Mitochondria are cellular energy powerhouses that play important roles in maintaining cell survival, cell death and cellular metabolic homeostasis. Timely removal of damaged mitochondria via autophagy (mitophagy) is thus critical for cellular homeostasis and function. Mitochondria are reticular organelles that have high plasticity for their dynamic structures and constantly undergo fission and fusion as well as movement through the cytoskeleton. In this review, we discuss the most recent progress on the molecular mechanisms and roles of mitochondrial fission/fusion and mitochondrial motility in mitophagy. We also discuss multiple pathways leading to the quality control of mitochondria in addition to the traditional mitophagy pathway under different conditions.

Nrf2 promotes the development of fibrosis and tumorigenesis in mice with defective hepatic autophagy

BACKGROUND & AIMS Autophagy is an intracellular lysosomal degradation process that plays an important role in regulating normal physiological functions of the liver. The purpose of the present study was to investigate the mechanism(s) by which the loss of hepatic autophagy leads to liver inflammation, fibrosis and tumorigenesis. METHODS Hepatocyte-specific Atg5 knockout mice were generated by crossing Atg5 Flox/Flox mice with albumin Cre mice. These mice were also crossed with Nrf2 knockout mice to generate Atg5 Flox/Flox, Albumin Cre(+)/Nrf2(-/-) double knockout mice. These mice were housed for various time points up to 15 months, and blood and liver tissues were harvested for biochemical and histological analysis. RESULTS Hepatocyte-specific deletion of Atg5 resulted in increased apoptosis, inflammation and fibrosis in the liver. Increased apoptosis in hepatocyte-specific Atg5 knockout mice was likely due to accumulation of aberrant polyubiquitinated proteins (proteotoxicity) and disruption of the homeostasis of pro-and anti-apoptotic proteins. All of these pathological changes started as early as one month and persisted for 12-15 months. At 9-15 months of age, these mice also developed hepatocellular adenomas. Interestingly, deletion of Nrf2 in Atg5 liver-specific knockout mice markedly abolished these pathological changes, indicating a key role for this transcription factor in the mechanism of hepatic pathology. CONCLUSIONS Our results provide genetic evidence that loss of autophagy in hepatocytes causes cell death resulting in liver inflammation, fibrosis and tumorigenesis. We also demonstrate that persistent activation of Nrf2 is critical for liver inflammation, fibrosis and eventual tumorigenesis that occur in mice with defects in hepatocyte autophagy.

Role of p62/SQSTM1 in liver physiology and pathogenesis.

p62/sequestosome-1/A170/ZIP (hereafter referred to as p62) is a scaffold protein that has multiple functions, such as signal transduction, cell proliferation, cell survival, cell death, inflammation, tumourigenesis and oxidative stress response. While p62 is an autophagy substrate and is degraded by autophagy, p62 serves as an autophagy receptor for selective autophagic clearance of protein aggregates and organelles. Moreover, p62 functions as a signalling hub for various signalling pathways, including NF-κB, Nrf2 and mTOR. In this review, we discuss the pathophysiological role of p62 in the liver, including formation of hepatic inclusion bodies, cholestasis, obesity, insulin resistance, liver cell death and tumourigenesis.

Zonated induction of autophagy and mitochondrial spheroids limits acetaminophen-induced necrosis in the liver

Acetaminophen (APAP) overdose is the most frequent cause of acute liver failure in the US and many western countries. It is well known that APAP induces mitochondrial damage to trigger centrilobular necrosis. Emerging evidence suggests that autophagic removal of damaged mitochondria may protect against APAP-induced liver injury. Electron and confocal microscopy analysis of liver tissues revealed that APAP overdose triggers unique biochemical and pathological zonated changes in the mouse liver, which includes necrosis (zone 1), mitochondrial spheroid formation (zone 2), autophagy (zone 3) and mitochondrial biogenesis (zone 4). In this graphic review, we discuss the role of autophagy/mitophagy in limiting the expansion of necrosis and promoting mitochondrial biogenesis and liver regeneration for the recovery of APAP-induced liver injury. We also discuss possible mechanisms that could be involved in regulating APAP-induced autophagy/mitophagy and the formation of mitochondrial spheroids.

Targeting autophagy for the treatment of liver diseases

Autophagy is a lysosomal degradation pathway that can degrade bulk cytoplasm and superfluous or damaged organelles, such as mitochondria, to maintain cellular homeostasis. It is now known that dysregulation of autophagy can cause pathogenesis of numerous human diseases. Here, we discuss the critical roles that autophagy plays in the pathogenesis of liver diseases such as non-alcoholic and alcoholic fatty liver, drug-induced liver injury, protein aggregate-related liver diseases, viral hepatitis, fibrosis, aging and liver cancer. In particular, we discuss the emerging therapeutic potential by pharmacological modulation of autophagy for these liver diseases.

Fibroblast growth factor 15 deficiency impairs liver regeneration in mice

Fibroblast growth factor (FGF) 15 (human homolog, FGF19) is an endocrine FGF highly expressed in the small intestine of mice. Emerging evidence suggests that FGF15 is critical for regulating hepatic functions; however, the role of FGF15 in liver regeneration is unclear. This study assessed whether liver regeneration is altered in FGF15 knockout (KO) mice following 2/3 partial hepatectomy (PHx). The results showed that FGF15 KO mice had marked mortality, with the survival rate influenced by genetic background. Compared with wild-type mice, the KO mice displayed extensive liver necrosis and marked elevation of serum bile acids and bilirubin. Furthermore, hepatocyte proliferation was reduced in the KO mice because of impaired cell cycle progression. After PHx, the KO mice had weaker activation of signaling pathways that are important for liver regeneration, including signal transducer and activator of transcription 3, nuclear factor-κB, and mitogen-activated protein kinase. Examination of the KO mice at early time points after PHx revealed a reduced and/or delayed induction of immediate-early response genes, including growth-control transcription factors that are critical for liver regeneration. In conclusion, the results suggest that FGF15 deficiency severely impairs liver regeneration in mice after PHx. The underlying mechanism is likely the result of disrupted bile acid homeostasis and impaired priming of hepatocyte proliferation.

Parkin regulates mitophagy and mitochondrial function to protect against alcohol-induced liver injury and steatosis in mice.

Alcoholic liver disease claims two million lives per year. We previously reported that autophagy protected against alcohol-induced liver injury and steatosis by removing damaged mitochondria. However, the mechanisms for removal of these mitochondria are unknown. Parkin is an evolutionarily conserved E3 ligase that is recruited to damaged mitochondria to initiate ubiquitination of mitochondrial outer membrane proteins and subsequent mitochondrial degradation by mitophagy. In addition to its role in mitophagy, Parkin has been shown to have other roles in maintaining mitochondrial function. We investigated whether Parkin protected against alcohol-induced liver injury and steatosis using wild-type (WT) and Parkin knockout (KO) mice treated with alcohol by the acute-binge and Gao-binge (chronic plus acute-binge) models. We found that Parkin protected against liver injury in both alcohol models, likely because of Parkins role in maintaining a population of healthy mitochondria. Alcohol caused greater mitochondrial damage and oxidative stress in Parkin KO livers compared with WT livers. After alcohol treatment, Parkin KO mice had severely swollen and damaged mitochondria that lacked cristae, which were not seen in WT mice. Furthermore, Parkin KO mice had decreased mitophagy, β-oxidation, mitochondrial respiration, and cytochrome c oxidase activity after acute alcohol treatment compared with WT mice. Interestingly, liver mitochondria seemed able to adapt to alcohol treatment, but Parkin KO mouse liver mitochondria had less capacity to adapt to Gao-binge treatment compared with WT mouse liver mitochondria. Overall, our findings indicate that Parkin is an important mediator of protection against alcohol-induced mitochondrial damage, steatosis, and liver injury.

New advances in molecular mechanisms and emerging therapeutic targets in alcoholic liver diseases

Alcoholic liver disease is a major health problem in the United States and worldwide. Chronic alcohol consumption can cause steatosis, inflammation, fibrosis, cirrhosis and even liver cancer. Significant progress has been made to understand key events and molecular players for the onset and progression of alcoholic liver disease from both experimental and clinical alcohol studies. No successful treatments are currently available for treating alcoholic liver disease; therefore, development of novel pathophysiological-targeted therapies is urgently needed. This review summarizes the recent progress on animal models used to study alcoholic liver disease and the detrimental factors that contribute to alcoholic liver disease pathogenesis including miRNAs, S-adenosylmethionine, Zinc deficiency, cytosolic lipin-1β, IRF3-mediated apoptosis, RIP3-mediated necrosis and hepcidin. In addition, we summarize emerging adaptive protective effects induced by alcohol to attenuate alcohol-induced liver pathogenesis including FoxO3, IL-22, autophagy and nuclear lipin-1α.

Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice

BACKGROUND & AIMS Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in the US and many other countries. Metabolism of APAP results in formation of APAP protein adducts (APAP-AD) in hepatocytes and triggers mitochondrial dysfunction and necrosis. However, the mechanisms for how APAP-AD are removed from hepatocytes remain unknown. METHODS Mice or primary hepatocytes were treated with APAP. APAP-AD were determined by immunoblot, immunostaining and high pressure liquid chomatography with electrochemical detection analysis. RESULTS We found that APAP-AD were detected at 1h, peaked at approximately 2h, declined at 6h and almost full removed at 24h post treatment with APAP in mouse livers and in primary mouse hepatocytes. APAP-AD displayed a punctate pattern and were colocalized with GFP-LC3 positive autophagosomes and Lamp1 positive lysosomes in APAP-treated primary hepatocytes. Moreover, isolated autophagosomes and autolysosomes from APAP-treated mouse livers contained APAP-AD, suggesting autophagy may selectively remove APAP-AD. APAP-AD were detected in both detergent soluble and insoluble pools in APAP-treated mouse livers and hepatocytes. More importantly, pharmacological inhibition of autophagy by leupeptin or chloroquine increased whereas induction of autophagy by Torin 1 decreased serum APAP-AD levels in APAP-treated mice, which correlated with alanine aminotransferase levels and liver necrosis. Furthermore, SQSTM1/p62, an autophagy receptor protein, was recruited to APAP-AD. Adenovirus-mediated shRNA knockdown of SQSTM1/p62 led to increased APAP-AD and necrosis in primary hepatocytes. CONCLUSIONS Our data indicate that APAP-AD are removed though selective autophagy. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury. LAY SUMMARY Acetaminophen overdose can form acetaminophen protein adducts and mitochondria damage in hepatocytes resulting in liver injury. Activation of autophagy-lysosomal degradation pathway can help to remove acetaminophen protein adducts. Pharmacological induction of autophagy may be a novel promising approach for treating APAP-induced liver injury.

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

Background: Parkin is required for mitophagy in cultured cells, but its role in liver injury is not known. Results: APAP-induced mitophagy was impaired in acute Parkin knockdown mouse livers but not whole body Parkin knock-out mice. Conclusion: Chronic, but not acute, loss of Parkin protects against APAP-induced liver injury by multiple mechanisms independent of mitophagy. Significance: This study revealed a novel role of Parkin in APAP-induced liver injury. 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.