Mitochondrial quality control protects photoreceptors against oxidative stress in the H2O2-induced models of retinal degeneration diseases

Retinal degeneration diseases (RDDs) are common and devastating eye diseases characterized by the degeneration of photoreceptors, which are highly associated with oxidative stress. Previous studies reported that mitochondrial dysfunction is associated with various neurodegenerative diseases. However, the role of mitochondrial proteostasis mainly regulated by mitophagy and mitochondrial unfolded protein response (mtUPR) in RDDs is unclear. We hypothesized that the mitochondrial proteostasis is neuroprotective against oxidative injury in RDDs. In this study, the data from our hydrogen peroxide (H₂O₂)-treated mouse retinal cone cell line (661w) model of RDDs showed that nicotinamide riboside (NR)-activated mitophagy increased the expression of LC3B II and PINK1, and promoted the co-localization of LC3 and mitochondria, as well as PINK1 and Parkin in the H₂O₂-treated 661w cells. However, the NR-induced mitophagy was remarkably reversed by chloroquine (CQ) and cyclosporine A (CsA), mitophagic inhibitors. In addition, doxycycline (DOX), an inducer of mtUPR, up-regulated the expression of HSP60 and CHOP, the key proteins of mtUPR. Activation of both mitophagy and mtUPR increased the cell viability and reduced the level of apoptosis and oxidative damage in the H₂O₂-treated 661w cells. Furthermore, both mitophagy and mtUPR played a protective effect on mitochondria by increasing mitochondrial membrane potential and maintaining mitochondrial mass. By contrast, the inhibition of mitophagy by CQ or CsA reversed the beneficial effect of mitophagy in the H₂O₂-treated 661w cells. Together, our study suggests that the mitophagy and mtUPR pathways may serve as new therapeutic targets to delay the progression of RDDs through enhancing mitochondrial proteostasis.Subject terms: Cell death, Diseases     

Bacterial extracellular vesicles induced oxidative stress and mitophagy through mTOR pathways in colon cancer cells,HT-29: Implications for bioactivity

Bacterial-extracellular-vesicles (BEVs) derived from Escherichia coli, strain-A5922, were used as a therapeutic tool to treat colon cancer cells, HT-29. BEVs induced oxidative stress, and observed mitochondrial autophagy, known as mitophagy, were crucial in initiation of treatment. The mitophagy, induced by the BEVs in HT-29 cells, produced adenocarcinomic cytotoxicity, and stopped the cells growth. The trigger for mitophagy, and an increase in productions of reactive oxygen species led to cellular oxidative stress, that eventually led to cells death. A reduction in the mitochondrial membrane potential, and an increase in the PINK1 expressions confirmed the oxidative stress involvements. The BEVs triggered cytotoxicity, and mitophagy in the HT-29 carcinoid cells, channelized through the Akt/mTOR pathways connecting the cellular oxidative stress, effectively played its part to cause cells death. These findings substantiated the BEVs' potential as a plausible tool for treating, and possibly preventing the colorectal cancer.     

The role of mitochondrial cardiolipin in heart function and its implication in cardiac disease

Mitochondria play an essential role in the energy metabolism of the heart. Many of the essential functions are associated with mitochondrial membranes and oxidative phosphorylation driven by the respiratory chain. Mitochondrial membranes are unique in the cell as they contain the phospholipid cardiolipin. The important role of cardiolipin in cardiovascular health is highlighted by several cardiac diseases, in which cardiolipin plays a fundamental role. Barth syndrome, Sengers syndrome, and Dilated cardiomyopathy with ataxia (DCMA) are genetic disorders, which affect cardiolipin biosynthesis. Other cardiovascular diseases including ischemia/reperfusion injury and heart failure are also associated with changes in the cardiolipin pool. Here, we summarize molecular functions of cardiolipin in mitochondrial biogenesis and morphology. We highlight the role of cardiolipin for the respiratory chain, metabolite carriers, and mitochondrial metabolism and describe links to apoptosis and mitochondria specific autophagy (mitophagy) with possible implications in cardiac disease.     

A critical role of superoxide anion in selenite-induced mitophagic cell death

Mitochondria, which are a major source of intracellular reactive oxygen species (ROS), are extremely vulnerable to oxidative stress. We recently reported that selenite treatment of various glioma cells induced a non-apoptotic cell death accompanied by excessive mitophagy (selective autophagy of damaged mitochondria). Examination of various ROS revealed that the superoxide anion played a key role in selenite-induced mitochondrial damage, mitophagy and cell death. Treatment with superoxide generators (diquat and paraquat) was sufficient to trigger mitophagy in glioma cells. Small interfering RNA-mediated knockdown of ATG6 or ATG7 attenuated selenite-induced mitophagy and cell death, demonstrating that the mitophagic pathway contributes to selenite-induced cell death. The effect of selenite in glioma cells may thus provide an example of superoxide-mediated mitophagic cell death, i.e., cell death caused by excessive mitophagy.     

A critical role of superoxide anion in selenite-induced mitophagic cell death

Mitochondria, which are a major source of intracellular reactive oxygen species (ROS), are extremely vulnerable to oxidative stress. We recently reported that selenite treatment of various glioma cells induced a non-apoptotic cell death accompanied by excessive mitophagy (selective autophagy of damaged mitochondria). Examination of various ROS revealed that the superoxide anion played a key role in selenite-induced mitochondrial damage, mitophagy and cell death. Treatment with superoxide generators (diquat and paraquat) was sufficient to trigger mitophagy in glioma cells. Small interfering RNA-mediated knockdown of ATG6 or ATG7 attenuated selenite-induced mitophagy and cell death, demonstrating that the mitophagic pathway contributes to selenite-induced cell death. The effect of selenite in glioma cells may thus provide an example of superoxide-mediated mitophagic cell death, i.e., cell death caused by excessive mitophagy.

Addendum to: Kim EH, Sohn S, Kwon HJ, Kim SU, Kim MJ, Lee SJ, Choi KS. Sodium selenite induces superoxide-mediated mitochondrial damage and subsequent autophagic cell death in malignant glioma cells. Cancer Res 2007; 67:6314-24     

Upregulated autophagy protects cardiomyocytes from oxidative stress-induced toxicity

Autophagy is a cellular self-digestion process that mediates protein quality control and serves to protect against neurodegenerative disorders, infections, inflammatory diseases and cancer. Current evidence suggests that autophagy can selectively remove damaged organelles such as the mitochondria. Mitochondria-induced oxidative stress has been shown to play a major role in a wide range of pathologies in several organs, including the heart. Few studies have investigated whether enhanced autophagy can offer protection against mitochondrially-generated oxidative stress. We induced mitochondrial stress in cardiomyocytes using antimycin A (AMA), which increased mitochondrial superoxide generation, decreased mitochondrial membrane potential and depressed cellular respiration. In addition, AMA augmented nuclear DNA oxidation and cell death in cardiomyocytes. Interestingly, although oxidative stress has been proposed to induce autophagy, treatment with AMA did not result in stimulation of autophagy or mitophagy in cardiomyocytes. Our results showed that the MTOR inhibitor rapamycin induced autophagy, promoted mitochondrial clearance and protected cardiomyocytes from the cytotoxic effects of AMA, as assessed by apoptotic marker activation and viability assays in both mouse atrial HL-1 cardiomyocytes and human ventricular AC16 cells. Importantly, rapamycin improved mitochondrial function, as determined by cellular respiration, mitochondrial membrane potential and morphology analysis. Furthermore, autophagy induction by rapamycin suppressed the accumulation of ubiquitinylated proteins induced by AMA. Inhibition of rapamycin-induced autophagy by pharmacological or genetic interventions attenuated the cytoprotective effects of rapamycin against AMA. We propose that rapamycin offers cytoprotection against oxidative stress by a combined approach of removing dysfunctional mitochondria as well as by degrading damaged, ubiquitinated proteins. We conclude that autophagy induction by rapamycin could be utilized as a potential therapeutic strategy against oxidative stress-mediated damage in cardiomyocytes.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

Cardiolipin in Central Nervous System Physiology and Pathology

Cardiolipin, an anionic phospholipid found primarily in the inner mitochondrial membrane, has many well-defined roles within the peripheral tissues, including the maintenance of mitochondrial membrane fluidity and the regulation of mitochondrial functions. Within the central nervous system (CNS), cardiolipin is found within both neuronal and non-neuronal glial cells, where it regulates metabolic processes, supports mitochondrial functions, and promotes brain cell viability. Furthermore, cardiolipin has been shown to act as an elimination signal and participate in programmed cell death by apoptosis of both neurons and glia. Since cardiolipin is associated with regulating brain homeostasis, the modification of its structure, or even a decrease in the overall levels of cardiolipin, can result in mitochondrial dysfunction, which is a characteristic feature of many diseases. In this review, we outline the various functions of cardiolipin within the cells of the CNS, including neurons, astrocytes, microglia, and oligodendrocytes. In addition, we discuss the role cardiolipin may play in the pathogenesis of the neurodegenerative disorders Alzheimer’s disease and Parkinson’s disease, as well as traumatic brain injury.     

4-HPR-mediated leukemia cell cytotoxicity is triggered by ceramide-induced mitochondrial oxidative stress and is regulated downstream by Bcl-2

We have previously reported that, in leukemia cells, the cytotoxicity of the anticancer agent N-(4-hydroxyphenyl)retinamide (4-HPR) is mediated by mitochondria-derived reactive oxygen species (ROS) and cardiolipin peroxidation. Here, we have analyzed at greater depth the 4-HPR-triggered molecular events, demonstrating that 4-HPR induces an early (15 min) increase in ceramide levels by sphingomyelin hydrolysis and later (from 1 h) by de novo synthesis. Using specific inhibitors of both pathways, we demonstrate that ceramide accumulation is responsible for early ROS generation, which act as apoptotic signalling intermediates leading to conformational activation of Bak and Bax, loss of mitochondrial membrane potential (ΔΨm), mitochondrial membrane permeabilization (MMP) and cell death. Enforced expression of Bcl-2 has no effect on 4-HPR-induced oxidative stress, but notably prevents mitochondrial alterations and apoptosis, indicating that Bcl-2 functions by regulating events downstream of ROS generation. In conclusion, our study delineates for the fist time the sequence and timing of the principal events induced by 4-HPR in leukemia cells and points to the potential use of modulators of ceramide metabolism as enhancers in 4-HPR-based therapies.     

A mitophagic response to iron overload-induced oxidative damage associated with the PINK1/Parkin pathway in pancreatic beta cells

BackgroundIron overload can result in a disorder in glucose metabolism. However, the underlining mechanism through which iron overload induces beta cell death remains unknown.MethodsAccording to the concentration of ferric ammonium citrate (FAC) and N-acetylcysteine, INS-1 cells were randomly divided into four groups: normal control (FAC 0 μM) group, FAC 80 μM group, FAC 160 μM group, FAC 160μM + NAC group. Cell proliferation was assessed by Cell Counting Kit-8. Reactive oxygen species (ROS) level was further evaluated using flow cytometer with a fluorescent probe. The mitochondrial membrane potential was detected by JC-1 kit, and transmission electron microscopy was used to observe the mitochondrial changes. The related protein expressions were detected by western bolt to evaluate mitophagy status.ResultsIt was shown that FAC treatment decreased INS-1 cell viability in vitro, resulted in a decline in mitochondrial membrane potential, increased oxidative stress level and suppressed mitophagy. Furthermore, these effects could be alleviated by the ROS scavenger.ConclusionsWe proved that increased iron overload primarily increased oxidative stress and further suppressed mitophagy via PTEN-induced putative kinase 1/Parkin pathway, resulting in cytotoxicity in INS-1 cells.     

Caveolin-1 knockdown increases the therapeutic sensitivity of lung cancer to cisplatin-induced apoptosis by repressing Parkin-related mitophagy and activating the ROCK1 pathway

Chemotherapy is the first-line treatment option for patients with lung cancer. However, therapeutic resistance occurs through an incompletely understood mechanism. Our research wants to investigate the influence of Caveolin-1 (Cav-1) on the therapeutic sensitivity of lung cancer in vitro. Results in this study demonstrated that Cav-1 levels were markedly inhibited in A549 lung cancer cells after exposure to cisplatin. Knockdown of caveolin further enhanced cisplatin-triggered cancer death in A549 cells. The functional investigation demonstrated that Cav-1 inhibition amplified the mitochondrial stress signaling induced by cisplatin, as evidenced by the mitochondrial reactive oxygen species burst, cellular metabolic disruption, mitochondrial membrane potential reduction, and mitochondrial caspase-9-related apoptosis activation. At the molecular level, cav-1 augmented cisplatin-mediated mitochondrial damage by inhibiting Parkin-related mitochondrial autophagy. Mitophagy activation effectively attenuated the promotive impact of Cav-1 knockdown on mitochondrial damage and cell death. Furthermore, our data indicated that Cav-1 affected Parkin-related mitophagy by activating the Rho-associated coiled-coil kinase 1 (ROCK1) pathway; inhibition of the ROCK1 axis prevented cav-1 knockdown-mediated cell death and mitochondrial damage. Taken together, our results provide ample data illuminate the necessary action exerted by Cav-1 on affecting cisplatin-related therapeutic resistance. Silencing of Cav-1 inhibited Parkin-related mitophagy, thus amplifying cisplatin-mediated mitochondrial apoptotic signaling. This finding identifies the Cav-1/ROCK1/Parkin/mitophagy axis as a potential target to overcome cisplatin-related resistance in lung cancer cells.     

Cholesterol and peroxidized cardiolipin in mitochondrial membrane properties,permeabilization and cell death

Mitochondria are known to actively regulate cell death with the final phenotype of demise being determined by the metabolic and energetic status of the cell. Mitochondrial membrane permeabilization (MMP) is a critical event in cell death, as it regulates the degree of mitochondrial dysfunction and the release of intermembrane proteins that function in the activation and assembly of caspases. In addition to the crucial role of proapoptotic members of the Bcl-2 family, the lipid composition of the mitochondrial membranes is increasingly recognized to modulate MMP and hence cell death. The unphysiological accumulation of cholesterol in mitochondrial membranes regulates their physical properties, facilitating or impairing MMP during Bax and death ligand-induced cell death depending on the level of mitochondrial GSH (mGSH), which in turn regulates the oxidation status of cardiolipin. Cholesterol-mediated mGSH depletion stimulates TNF-induced reactive oxygen species and subsequent cardiolipin peroxidation, which destabilizes the lipid bilayer and potentiates Bax-induced membrane permeabilization. These data suggest that the balance of mitochondrial cholesterol to peroxidized cardiolipin regulates mitochondrial membrane properties and permeabilization, emerging as a rheostat in cell death.     

Mitophagy,a potential therapeutic target for stroke

Mitochondria autophagy, termed as mitophagy, is a mechanism of specific autophagic elimination of mitochondria. Mitophagy controls the quality and the number of mitochondria, eliminating dysfunctional or excessive mitochondria that can generate reactive oxygen species (ROS) and cause cell death. Mitochondria are centrally implicated in neuron and tissue injury after stroke, due to the function of supplying adenosine triphosphate (ATP) to the tissue, regulating oxidative metabolism during the pathologic process, and contribution to apoptotic cell death after stroke. As a catabolic mechanism, mitophagy links numbers of a complex network of mitochondria, and affects mitochondrial dynamic process, fusion and fission, reducing mitochondrial production of ROS, mediated by the mitochondrial permeability transition pore (MPTP). The precise nature of mitophagy’s involvement in stroke, and its underlying molecular mechanisms, have yet to be fully clarified. This review aims to provide a comprehensive overview of the integration of mitochondria with mitophagy, also to introduce and discuss recent advances in the understanding of the potential role, and possible signaling pathway, of mitophagy in the pathological processes of both hemorrhagic and ischemic stroke. The author also provides evidence to explain the dual role of mitophagy in stroke.     

Cardiolipin remodeling by TAZ/tafazzin is selectively required for the initiation of mitophagy

Tafazzin (TAZ) is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of TAZ cause Barth syndrome, which is characterized by mitochondrial dysfunction and dilated cardiomyopathy, leading to premature death. However, the molecular mechanisms underlying the cause of mitochondrial dysfunction in Barth syndrome remain poorly understood. Here we investigated the role of TAZ in regulating mitochondrial function and mitophagy. Using primary mouse embryonic fibroblasts (MEFs) with doxycycline-inducible knockdown of Taz, we showed that TAZ deficiency in MEFs caused defective mitophagosome biogenesis, but not other autophagic processes. Consistent with a key role of mitophagy in mitochondria quality control, TAZ deficiency in MEFs also led to impaired oxidative phosphorylation and severe oxidative stress. Together, these findings provide key insights on mitochondrial dysfunction in Barth syndrome, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for this lethal condition.     

Oxidative stress,mitochondrial bioenergetics,and cardiolipin in aging

Aging is a natural, complex, and multifactorial biological process associated with impairment of bioenergetic function, increased oxidative stress, attenuated ability to respond to stresses, and increased risk of contracting age-associated diseases. Oxidative stress is widely thought to underpin many aging processes. The mitochondrion, the powerhouse of the cell, is considered the most important cellular organelle to contribute to the aging process, mainly through respiratory chain dysfunction and formation of reactive oxygen species, leading to damage to mitochondrial proteins, lipids, and mitochondrial DNA. Cardiolipin, a phospholipid located at the level of the inner mitochondrial membrane, is known to be intimately involved in several mitochondrial bioenergetic processes as well as mitochondrial-dependent steps in apoptosis and mitochondrial membrane stability and dynamics. Alterations to cardiolipin structure, content, and acyl chain composition have been associated with mitochondrial dysfunction in multiple tissues in several physiopathological conditions and aging. In this review, we discuss several aspects of mitochondrial bioenergetic alterations in aging and the role played by reactive oxygen species and cardiolipin in these alterations.     

ATAD3B is a mitophagy receptor mediating clearance of oxidative stress‐induced damaged mitochondrial DNA

Mitochondrial DNA (mtDNA) encodes several key components of respiratory chain complexes that produce cellular energy through oxidative phosphorylation. mtDNA is vulnerable to damage under various physiological stresses, especially oxidative stress. mtDNA damage leads to mitochondrial dysfunction, and dysfunctional mitochondria can be removed by mitophagy, an essential process in cellular homeostasis. However, how damaged mtDNA is selectively cleared from the cell, and how damaged mtDNA triggers mitophagy, remain mostly unknown. Here, we identified a novel mitophagy receptor, ATAD3B, which is specifically expressed in primates. ATAD3B contains a LIR motif that binds to LC3 and promotes oxidative stress‐induced mitophagy in a PINK1‐independent manner, thus promoting the clearance of damaged mtDNA induced by oxidative stress. Under normal conditions, ATAD3B hetero‐oligomerizes with ATAD3A, thus promoting the targeting of the C‐terminal region of ATAD3B to the mitochondrial intermembrane space. Oxidative stress‐induced mtDNA damage or mtDNA depletion reduces ATAD3B‐ATAD3A hetero‐oligomerization and leads to exposure of the ATAD3B C‐terminus at the mitochondrial outer membrane and subsequent recruitment of LC3 for initiating mitophagy. Furthermore, ATAD3B is little expressed in m.3243A > G mutated cells and MELAS patient fibroblasts showing endogenous oxidative stress, and ATAD3B re‐expression promotes the clearance of m.3243A > G mutated mtDNA. Our findings uncover a new pathway to selectively remove damaged mtDNA and reveal that increasing ATAD3B activity is a potential therapeutic approach for mitochondrial diseases.     

Increase in cytosolic Ca2+ levels through the activation of non-selective cation channels induced by oxidative stress causes mitochondrial depolarization leading to apoptosis-like death in Leishmania donovani promastigotes

Reactive oxygen species are important regulators of protozoal infection. Promastigotes of Leishmania donovani, the causative agent of Kala-azar, undergo an apoptosis-like death upon exposure to H2O2. The present study shows that upon activation of death response by H2O2, a dose- and time-dependent loss of mitochondrial membrane potential occurs. This loss is accompanied by a depletion of cellular glutathione, but cardiolipin content or thiol oxidation status remains unchanged. ATP levels are reduced within the first 60 min of exposure as a result of mitochondrial membrane potential loss. A tight link exists between changes in cytosolic Ca2+ homeostasis and collapse of the mitochondrial membrane potential, but the dissipation of the potential is independent of elevation of cytosolic Na+ and mitochondrial Ca2+. Partial inhibition of cytosolic Ca2+ increase achieved by chelating extracellular or intracellular Ca2+ by the use of appropriate agents resulted in significant rescue of the fall of the mitochondrial membrane potential and apoptosis-like death. It is further demonstrated that the increase in cytosolic Ca2+ is an additive result of release of Ca2+ from intracellular stores as well as by influx of extracellular Ca2+ through flufenamic acid-sensitive non-selective cation channels; contribution of the latter was larger. Mitochondrial changes do not involve opening of the mitochondrial transition pore as cyclosporin A is unable to prevent mitochondrial membrane potential loss. An antioxidant like N-acetylcysteine is able to inhibit the fall of the mitochondrial membrane potential and prevent apoptosis-like death. Together, these findings show the importance of non-selective cation channels in regulating the response of L. donovani promastigotes to oxidative stress that triggers downstream signaling cascades leading to apoptosis-like death.     

Protective effect of kombucha tea against tertiary butyl hydroperoxide induced cytotoxicity and cell death in murine hepatocytes

Kombucha (KT), a fermented black tea (BT), is known to have many beneficial properties. In the present study, antioxidant property of KT has been investigated against tertiary butyl hydroperoxide (TBHP) induced cytotoxicity using murine hepatocytes. TBHP, a reactive oxygen species inducer, causes oxidative stress resulting in organ pathophysiology. Exposure to TBHP caused a reduction in cell viability, increased membrane leakage and disturbed the intra-cellular antioxidant machineries in hepatocytes. TBHP exposure disrupted mitochondrial membrane potential and induced apoptosis as evidenced by flow cytometric analyses. KT treatment, however, counteracted the changes in mitochondrial membrane potential and prevented apoptotic cell death of the hepatocytes. BT treatment also reverted TBHP induced hepatotoxicity, however KT was found to be more efficient. This may be due to the formation of antioxidant molecules like D-saccharic acid-1,4-lactone (DSL) during fermentation process and are absent in BT. Moreover, the radical scavenging activities of KT were found to be higher than BT. Results of the study showed that KT has the potential to ameliorate TBHP induced oxidative insult and cell death in murine hepatocytes more effectively than BT.     

Liraglutide alleviates H2O2-induced retinal ganglion cells injury by inhibiting autophagy through mitochondrial pathways

Retinal ganglion cells (RGCs), which exist in the inner retina, are the retinal neurons which can be damaged in the early stage of diabetic retinopathy (DR). Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, exerts biological functions by binding the receptor (GLP-1R), the expression of which in RGC-5 cells was first shown by our team in 2012. It was reported that liraglutide prevented retinal neurodegeneration in diabetic subjects. However, the involvement of mechanisms such as autophagy and mitochondrial balance in liraglutide-induced retinal protection is unknown. Here, we aimed to investigate the protective effects of liraglutide and explore the potential mechanisms of liraglutide-induced retinal RGC protection. RGC-5 cells were treated with H₂O₂ and/or liraglutide. Cell viability was detected with the CCK-8 kit. The axon marker GAP43, autophagy and mitophagy indicators LC3A/B, Beclin-1, p62, Parkin, BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L) and the key regulator of mitochondrial biogenesis PGC-1α were examined via western blot analysis. Autophagy was also evaluated using the ImageXpress Micro XLS system and transmission electron microscopy (TEM). Reactive oxygen species (ROS), mitochondrial membrane potential and fluorescent staining for mitochondria were also measured using the ImageXpress Micro XLS system. Our results showed that pretreatment with liraglutide significantly prevented H₂O₂-induced cell viability decline, mitochondrial morphological deterioration and induction of autophagy, which appeared as increased expression of LC3 II/I and Beclin-1, along with p62 degradation. Moreover, liraglutide suppressed the H₂O₂-induced decline in GAP43 expression, thus protecting cells. However, rapamycin induced autophagy and blocked the protective process. Liraglutide also provided mitochondrial protection and appeared to alleviate H₂O₂-induced ROS overproduction and a decline in mitochondrial membrane potential, partially by promoting mitochondrial generation and attenuating mitophagy. In conclusion, liraglutide attenuates H₂O₂ induced RGC-5 cell injury by inhibiting autophagy through maintaining a balance between mitochondrial biogenesis and mitophagy.