Rapamycin Ameliorates Defects in Mitochondrial Fission and Mitophagy in Glioblastoma Cells

Glioblastoma (GBM) cells feature mitochondrial alterations, which are documented and quantified in the present study, by using ultrastructural morphometry. Mitochondrial impairment, which roughly occurs in half of the organelles, is shown to be related to mTOR overexpression and autophagy suppression. The novelty of the present study consists of detailing an mTOR-dependent mitophagy occlusion, along with suppression of mitochondrial fission. These phenomena contribute to explain the increase in altered mitochondria reported here. Administration of the mTOR inhibitor rapamycin rescues mitochondrial alterations. In detail, rapamycin induces the expression of genes promoting mitophagy (PINK1, PARKIN, ULK1, AMBRA1) and mitochondrial fission (FIS1, DRP1). This occurs along with over-expression of VPS34, an early gene placed upstream in the autophagy pathway. The topographic stoichiometry of proteins coded by these genes within mitochondria indicates that, a remarkable polarization of proteins involved in fission and mitophagy within mitochondria including LC3 takes place. Co-localization of these proteins within mitochondria, persists for weeks following rapamycin, which produces long-lasting mitochondrial plasticity. Thus, rapamycin restores mitochondrial status in GBM cells. These findings add novel evidence about mitochondria and GBM, while fostering a novel therapeutic approach to restore healthy mitochondria through mTOR inhibition.     

TNF-α Induces Mitophagy in Rheumatoid Arthritis Synovial Fibroblasts,and Mitophagy Inhibition Alleviates Synovitis in Collagen Antibody-Induced Arthritis

Mitophagy is a selective form of autophagy that removes damaged mitochondria. Increasing evidence indicates that dysregulated mitophagy is implicated in numerous autoimmune diseases, but the role of mitophagy in rheumatoid arthritis (RA) has not yet been reported. The aim of the present study was to determine the roles of mitophagy in patient-derived RA synovial fibroblasts (RASFs) and in the collagen antibody-induced arthritis mouse model. We measured the mitophagy marker PTEN-induced putative kinase 1 (PINK1) in RASFs treated with tumor necrosis factor-α (TNF-α) using Western blotting and immunofluorescence. Arthritis was induced in PINK1^−/− mice by intraperitoneal injection of an anti-type II collagen antibody cocktail and lipopolysaccharide. RA severity was assessed by histopathology. PINK1 expression and damaged mitochondria increased in TNF-α treated RASFs via increased intracellular levels of reactive oxygen species. PINK1 knockdown RASFs decreased cellular migration and invasion functions. In addition, PINK1^−/− mice with arthritis exhibited markedly reduced swelling and inflammation relative to wild-type mice with arthritis. Taken together, these findings suggest that regulation of PINK1 expression in RA could represent a potential therapeutic and diagnostic target for RA.     

Targetable Pathways for Alleviating Mitochondrial Dysfunction in Neurodegeneration of Metabolic and Non-Metabolic Diseases

Many neurodegenerative and inherited metabolic diseases frequently compromise nervous system function, and mitochondrial dysfunction and oxidative stress have been implicated as key events leading to neurodegeneration. Mitochondria are essential for neuronal function; however, these organelles are major sources of endogenous reactive oxygen species and are vulnerable targets for oxidative stress-induced damage. The brain is very susceptible to oxidative damage due to its high metabolic demand and low antioxidant defence systems, therefore minimal imbalances in the redox state can result in an oxidative environment that favours tissue damage and activates neuroinflammatory processes. Mitochondrial-associated molecular pathways are often compromised in the pathophysiology of neurodegeneration, including the parkin/PINK1, Nrf2, PGC1α, and PPARγ pathways. Impairments to these signalling pathways consequently effect the removal of dysfunctional mitochondria, which has been suggested as contributing to the development of neurodegeneration. Mitochondrial dysfunction prevention has become an attractive therapeutic target, and there are several molecular pathways that can be pharmacologically targeted to remove damaged mitochondria by inducing mitochondrial biogenesis or mitophagy, as well as increasing the antioxidant capacity of the brain, in order to alleviate mitochondrial dysfunction and prevent the development and progression of neurodegeneration in these disorders. Compounds such as natural polyphenolic compounds, bioactive quinones, and Nrf2 activators have been reported in the literature as novel therapeutic candidates capable of targeting defective mitochondrial pathways in order to improve mitochondrial function and reduce the severity of neurodegeneration in these disorders.     

Mitochondrial Dysfunction in Spinocerebellar Ataxia Type 3 Is Linked to VDAC1 Deubiquitination

Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado–Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.     

Downregulation of Mitochondrial TSPO Inhibits Mitophagy and Reduces Enucleation During Human Terminal Erythropoiesis

Translocator protein (TSPO) and voltage dependent anion channels (VDAC) are two proteins forming a macromolecular complex in the outer mitochondrial membrane that is involved in pleiotropic functions. Specifically, these proteins were described to regulate the clearance of damaged mitochondria by selective mitophagy in non-erythroid immortalized cell lines. Although it is well established that erythroblast maturation in mammals depends on organelle clearance, less is known about mechanisms regulating this clearance throughout terminal erythropoiesis. Here, we studied the effect of TSPO1 downregulation and the action of Ro5-4864, a drug ligand known to bind to the TSPO/VDAC complex interface, in ex vivo human terminal erythropoiesis. We found that both treatments delay mitochondrial clearance, a process associated with reduced levels of the PINK1 protein, which is a key protein triggering canonical mitophagy. We also observed that TSPO1 downregulation blocks erythroblast maturation at the orthochromatic stage, decreases the enucleation rate, and increases cell death. Interestingly, TSPO1 downregulation does not modify reactive oxygen species (ROS) production nor intracellular adenosine triphosphate (ATP) levels. Ro5-4864 treatment recapitulates these phenotypes, strongly suggesting an active role of the TSPO/VDAC complex in selective mitophagy throughout human erythropoiesis. The present study links the function of the TSPO/VDAC complex to the PINK1/Parkin-dependent mitophagy induction during terminal erythropoiesis, leading to the proper completion of erythroid maturation.     

DHA Protects Hepatocytes from Oxidative Injury through GPR120/ERK-Mediated Mitophagy

Oxidative stress occurs in a variety of clinical liver diseases and causes cellular damage and mitochondrial dysfunction. The clearance of damaged mitochondria by mitophagy may facilitate mitochondrial biogenesis and enhance cell survival. Although the supplementation of docosahexaenoic acid (DHA) has been recognized to relieve the symptoms of various liver diseases, the antioxidant effect of DHA in liver disease is still unclear. The purpose of our research was to investigate the antioxidant effect of DHA in the liver and the possible role of mitophagy in this. In vitro, H₂O₂-induced injury was caused in AML12 cells. The results showed that DHA repressed the level of reactive oxygen species (ROS) induced by H₂O₂ and stimulated the cellular antioxidation response. Most notably, DHA restored oxidative stress-impaired autophagic flux and promoted protective autophagy. In addition, PINK/Parkin-mediated mitophagy was activated by DHA in AML12 cells and alleviated mitochondrial dysfunction. The ERK1/2 signaling pathway was inhibited during oxidative stress but reactivated by DHA treatment. It was proven that the expression of ERK1/2 was involved in the regulation of mitophagy by the ERK1/2 inhibitor. We further proved these results in vivo. DHA effectively alleviated the liver oxidative damage caused by CCl₄ and enhanced antioxidation capacity; intriguingly, autophagy was also activated. In summary, our data demonstrated that DHA protected hepatocytes from oxidative damage through GPR120/ERK-mediated mitophagy.     

MoWhi2 Mediates Mitophagy to Regulate Conidiation and Pathogenesis in Magnaporthe oryzae

Mitophagy refers to the specific process of degrading mitochondria, which is an important physiological process to maintain the balance of mitochondrial quantity and quality in cells. At present, the mechanisms of mitophagy in pathogenic fungi remain unclear. Magnaporthe oryzae (Syn. Pyricularia oryzae), the causal agent of rice blast disease, is responsible for the most serious disease of rice. In M. oryzae, mitophagy occurs in the foot cells and invasive hyphae to promote conidiation and infection. In this study, fluorescent observations and immunoblot analyses showed that general stress response protein MoWhi2 is required for mitophagy in M. oryzae. In addition, the activation of the autophagy, pexophagy and cytoplasm-to-vacuole targeting (CVT) pathway upon nitrogen starvation was determined using the GFP-MoATG8, GFP-SRL and MoAPE1-GFP strains and the ΔMowhi2 mutant in these backgrounds. The results indicated that MoWhi2 is specifically required for mitophagy in M. oryzae. Further studies showed that mitophagy in the foot cells and invasive hyphae of the ΔMowhi2 was interrupted, leading to reduced conidiation and virulence in the ΔMowhi2 mutant. Taken together, we found that MoWhi2 contributes to conidiation and invasive growth by regulating mitophagy in M. oryzae.     

Colorectal Cancer Apoptosis Induced by Dietary δ-Valerobetaine Involves PINK1/Parkin Dependent-Mitophagy and SIRT3

Understanding the mechanisms of colorectal cancer progression is crucial in the setting of strategies for its prevention. δ-Valerobetaine (δVB) is an emerging dietary metabolite showing cytotoxic activity in colon cancer cells via autophagy and apoptosis. Here, we aimed to deepen current knowledge on the mechanism of δVB-induced colon cancer cell death by investigating the apoptotic cascade in colorectal adenocarcinoma SW480 and SW620 cells and evaluating the molecular players of mitochondrial dysfunction. Results indicated that δVB reduced cell viability in a time-dependent manner, reaching IC50 after 72 h of incubation with δVB 1.5 mM, and caused a G2/M cell cycle arrest with upregulation of cyclin A and cyclin B protein levels. The increased apoptotic cell rate occurred via caspase-3 activation with a concomitant loss in mitochondrial membrane potential and SIRT3 downregulation. Functional studies indicated that δVB activated mitochondrial apoptosis through PINK1/Parkin pathways, as upregulation of PINK1, Parkin, and LC3B protein levels was observed (p < 0.0001). Together, these findings support a critical role of PINK1/Parkin-mediated mitophagy in mitochondrial dysfunction and apoptosis induced by δVB in SW480 and SW620 colon cancer cells.     

Mitochondrial Dynamics and Mitophagy in Skeletal Muscle Health and Aging

The maintenance of mitochondrial integrity is critical for muscle health. Mitochondria, indeed, play vital roles in a wide range of cellular processes, including energy supply, Ca²⁺ homeostasis, retrograde signaling, cell death, and many others. All mitochondria-containing cells, including skeletal muscle cells, dispose of several pathways to maintain mitochondrial health, including mitochondrial biogenesis, mitochondrial-derived vesicles, mitochondrial dynamics (fusion and fission process shaping mitochondrial morphology), and mitophagy—the process in charge of the removal of mitochondria though autophagy. The loss of skeletal muscle mass (atrophy) is a major health problem worldwide, especially in older people. Currently, there is no treatment to counteract the progressive decline in skeletal muscle mass and strength that occurs with aging, a process termed sarcopenia. There is increasing data, including our own, suggesting that accumulation of dysfunctional mitochondria contributes to the development of sarcopenia. Impairments in mitochondrial dynamics and mitophagy were recently proposed to contribute to sarcopenia. This review summarizes the current state of knowledge on the role played by mitochondrial dynamics and mitophagy in skeletal muscle health and in the development of sarcopenia. We also highlight recent studies showing that enhancing mitophagy in skeletal muscle is a promising therapeutic target to prevent or even treat skeletal muscle dysfunction in the elderly.     

Proteomic Analyses Reveal that Sky1 Modulates Apoptosis and Mitophagy in Saccharomyces cerevisiae Cells Exposed to Cisplatin

Sky1 is the only member of the SR (Serine–Arginine) protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.     

S-Acetyl-Glutathione Attenuates Carbon Tetrachloride-Induced Liver Injury by Modulating Oxidative Imbalance and Inflammation

Liver fibrosis, depending on the stage of the disease, could lead to organ dysfunction and cirrhosis, and no effective treatment is actually available. Emergent proof supports a link between oxidative stress, liver fibrogenesis and mitochondrial dysfunction as molecular bases of the pathology. A valid approach to protect against the disease would be to replenish the endogenous antioxidants; thus, we investigated the protective mechanisms of the S-acetyl-glutathione (SAG), a glutathione (GSH) prodrug. Preliminary in vitro analyses were conducted on primary hepatic cells. SAG pre-treatment significantly protected against cytotoxicity induced by CCl4. Additionally, CCl4 induced a marked increase in AST and ALT levels, whereas SAG significantly reduced these levels, reaching values found in the control group. For the in vivo analyses, mice were administered twice a week with eight consecutive intraperitoneal injections of 1 mL/kg CCl4 (diluted at 1:10 in olive oil) to induce oxidative imbalance and liver inflammation. SAG (30 mg/kg) was administered orally for 8 weeks. SAG significantly restored SOD activity, GSH levels and GPx activity, while it strongly reduced GSSG levels, lipid peroxidation and H₂O₂ and ROS levels in the liver. Additionally, CCl4 induced a decrease in anti-oxidants, including Nrf2, HO-1 and NQO-1, which were restored by treatment with SAG. The increased oxidative stress characteristic on liver disfunction causes the impairment of mitophagy and accumulation of dysfunctional and damaged mitochondria. Our results showed the protective effect of SAG administration in restoring mitophagy, as shown by the increased PINK1 and Parkin expressions in livers exposed to CCl4 intoxication. Thus, the SAG administration showed anti-inflammatory effects decreasing pro-inflammatory cytokines TNF-α, IL-6, MCP-1 and IL-1β in both serum and liver, and suppressing the TLR4/NFkB pathway. SAG attenuated reduced fibrosis, collagen deposition, hepatocellular damage and organ dysfunction. In conclusion, our results suggest that SAG administration protects the liver from CCl4 intoxication by restoring the oxidative balance, ameliorating the impairment of mitophagy and leading to reduced inflammation.     

The Mitochondrial Kinase PINK1 in Diabetic Kidney Disease

Mitochondria are critical organelles that play a key role in cellular metabolism, survival, and homeostasis. Mitochondrial dysfunction has been implicated in the pathogenesis of diabetic kidney disease. The function of mitochondria is critically regulated by several mitochondrial protein kinases, including the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1). The focus of PINK1 research has been centered on neuronal diseases. Recent studies have revealed a close link between PINK1 and many other diseases including kidney diseases. This review will provide a concise summary of PINK1 and its regulation of mitochondrial function in health and disease. The physiological role of PINK1 in the major cells involved in diabetic kidney disease including proximal tubular cells and podocytes will also be summarized. Collectively, these studies suggested that targeting PINK1 may offer a promising alternative for the treatment of diabetic kidney disease.     

The Intracellular Distribution of the Small GTPase Rho5 and Its Dimeric Guanidine Nucleotide Exchange Factor Dck1/Lmo1 Determine Their Function in Oxidative Stress Response

Rho5, the yeast homolog of human Rac1, is a small GTPase which regulates the cell response to nutrient and oxidative stress by inducing mitophagy and apoptosis. It is activated by a dimeric GEF composed of the subunits Dck1 and Lmo1. Upon stress, all three proteins rapidly translocate from the cell surface (Rho5) and a diffuse cytosolic distribution (Dck1 and Lmo1) to mitochondria, with translocation of the GTPase depending on both GEF subunits. We here show that the latter associate with mitochondria independent from each other and from Rho5. The trapping of Dck1-GFP or GFP-Lmo1 to the mitochondrial surface by a specific nanobody fused to the transmembrane domain (TMD) of Fis1 results in a loss of function, mimicking the phenotypes of the respective gene deletions, dck1 or lmo1. Direct fusion of Rho5 to Fis1^TMD, i.e., permanent attachment to the mitochondria, also mimics the phenotypes of an rho5 deletion. Together, these data suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation in order to fulfill its function in the oxidative stress response. This notion is substantiated by the observation that strains carrying fusions of Rho5 to the cell wall integrity sensor Mid2, confining the GTPase to the plasma membrane, retained their function. We propose a model in which Rho5 activated at the plasma membrane represses the oxidative stress response under standard growth conditions. This repression is relieved upon its GEF-mediated translocation to mitochondria, thus triggering mitophagy and apoptosis.     

Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca²⁺ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca²⁺ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca²⁺ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca²⁺ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca²⁺ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.     

Metformin Restores Parkin-Mediated Mitophagy,Suppressed by Cytosolic p53

Metformin is known to alleviate hepatosteatosis by inducing 5’ adenosine monophosphate (AMP)-kinase-independent, sirtuin 1 (SIRT1)-mediated autophagy. Dysfunctional mitophagy in response to glucolipotoxicities might play an important role in hepatosteatosis. Here, we investigated the mechanism by which metformin induces mitophagy through restoration of the suppressed Parkin-mediated mitophagy. To this end, our ob/ob mice were divided into three groups: (1) ad libitum feeding of a standard chow diet; (2) intraperitoneal injections of metformin 300 mg/kg; and (3) 3 g/day caloric restriction (CR). HepG2 cells were treated with palmitate (PA) plus high glucose in the absence or presence of metformin. We detected enhanced mitophagy in ob/ob mice treated with metformin or CR, whereas mitochondrial spheroids were observed in mice fed ad libitum. Metabolically stressed ob/ob mice and PA-treated HepG2 cells showed an increase in expression of endoplasmic reticulum (ER) stress markers and cytosolic p53. Cytosolic p53 inhibited mitophagy by disturbing the mitochondrial translocation of Parkin, as demonstrated by immunoprecipitation. However, metformin decreased ER stress and p53 expression, resulting in induction of Parkin-mediated mitophagy. Furthermore, pifithrin-α, a specific inhibitor of p53, increased mitochondrial incorporation into autophagosomes. Taken together, these results indicate that metformin treatment facilitates Parkin-mediated mitophagy rather than mitochondrial spheroid formation by decreasing the inhibitory interaction with cytosolic p53 and increasing degradation of mitofusins.     

IL-25 Induced ROS-Mediated M2 Macrophage Polarization via AMPK-Associated Mitophagy

Interleukin (IL)-25 is a cytokine released by airway epithelial cells responding to pathogens. Excessive production of reactive oxygen species (ROS) leads to airway inflammation and remodeling in asthma. Mitochondria are the major source of ROS. After stress, defective mitochondria often undergo selective degradation, known as mitophagy. In this study, we examined the effects of IL-25 on ROS production and mitophagy and investigated the underlying mechanisms. The human monocyte cell line was pretreated with IL-25 at different time points. ROS production was measured by flow cytometry. The involvement of mitochondrial activity in the effects of IL-25 on ROS production and subsequent mitophagy was evaluated by enzyme-linked immunosorbent assay, Western blotting, and confocal microscopy. IL-25 stimulation alone induced ROS production and was suppressed by N-acetylcysteine, vitamin C, antimycin A, and MitoTEMPO. The activity of mitochondrial complex I and complex II/III and the levels of p-AMPK and the mitophagy-related proteins were increased by IL-25 stimulation. The CCL-22 secretion was increased by IL-25 stimulation and suppressed by mitophagy inhibitor treatment and PINK1 knockdown. The Th2-like cytokine IL-25 can induce ROS production, increase mitochondrial respiratory chain complex activity, subsequently activate AMPK, and induce mitophagy to stimulate M2 macrophage polarization in monocytes.