Mitophagy mediated by BNIP3 and NIX protects against ferroptosis by downregulating mitochondrial reactive oxygen species.

Mitophagy plays an important role in the maintenance of mitochondrial homeostasis and can be categorized into two types: ubiquitin-mediated and receptor-mediated pathways. During receptor-mediated mitophagy, mitophagy receptors facilitate mitophagy by tethering the isolation membrane to mitochondria. Although at least five outer mitochondrial membrane proteins have been identified as mitophagy receptors, their individual contribution and interrelationship remain unclear. Here, we show that HeLa cells lacking BNIP3 and NIX, two of the five receptors, exhibit a complete loss of mitophagy in various conditions. Conversely, cells deficient in the other three receptors show normal mitophagy. Using BNIP3/NIX double knockout (DKO) cells as a model, we reveal that mitophagy deficiency elevates mitochondrial reactive oxygen species (mtROS), which leads to activation of the Nrf2 antioxidant pathway. Notably, BNIP3/NIX DKO cells are highly sensitive to ferroptosis when Nrf2-driven antioxidant enzymes are compromised. Moreover, the sensitivity of BNIP3/NIX DKO cells is fully rescued upon the introduction of wild-type BNIP3 and NIX, but not the mutant forms incapable of facilitating mitophagy. Consequently, our results demonstrate that BNIP3 and NIX-mediated mitophagy plays a role in regulating mtROS levels and protects cells from ferroptosis.

Rigid but nonmetallic cranioplasty after pterional craniotomy: Technical note.

Background: Given the popularity of pterional craniotomy, numerous modifications have been made to prevent postoperative deformities. With the advent of titanium plates, fixation has become both simple and excellent. However, titanium plates can cause skin problems, infection, or cause skull growth to fail. Methods: To develop a simple, cost-effective, and esthetically satisfactory fixation method, without the use of non-metallic materials, six young and older patients underwent pterional craniotomy. CranioFix Absorbable clamps were used to fix the bone flap in the frontal and temporal regions such that the frontal part was in close contact with the skull. After fixation, the bone chips and bone dust were placed in the bone gap and fixed with fibrin glue. We measured the computed tomography values of the reconstructed area and thickness of the temporal profiles postoperatively over time. Results: Bone fusion was achieved in all patients by 1 year after surgery. Both the thickness of the temporalis muscle and the thickness of the temporal profile had changed within 2 mm as compared with the preoperative state. Conclusion: Our simple craniotomy technique, gentle tissue handling, and osteoplastic cranioplasty yielded satisfactory esthetic results and rigidness in pterional craniotomy.

Mitofissin: a novel mitochondrial fission protein that facilitates mitophagy.

ABBREVIATIONS: Atg: autophagy related; IMM: inner mitochondrial membrane; IMS: intermembrane space; PAS: phagophore assembly site; SAR: selective autophagy receptor.

Meet the authors: Tomoyuki Fukuda, Kentaro Furukawa, and Tomotake Kanki.

We talk to corresponding author Tomotake Kanki and co-first authors Tomoyuki Fukuda and Kentaro Furukawa about their paper "The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy" (this issue of Molecular Cell), their career paths, interests outside of their fields, and how they strike a work-life balance.

Midterm Clinical Outcomes for Deferred Coronary Revascularization on the Basis of Resting Full-Cycle Ratio and Fractional Flow Reserve Measurements.

The midterm prognosis of patients with deferred revascularization based on resting full-cycle ratio (RFR) or fractional flow reserve (FFR) is not well established. We investigated the midterm clinical outcomes of 137 consecutive patients with deferred revascularization of 177 coronary arteries based on RFR and FFR. Patients were classified into 3 groups (concordant normal, concordant abnormal, discordant FFR and RFR), using known cutoffs for FFR (≤0.80) and RFR (≤0.89). All-cause mortality occurred in 9 (6.6%) and major adverse cardiac events (MACEs) in 16 patients (11.7%). Concordant abnormal, age, body mass index (BMI), and current or history of cancer were associated with increased risks of all-cause mortality. In a multivariable model, current or history of cancer was significantly associated with all-cause death (hazard ratio [HR] 6.8, p = 0.02). Concordant abnormal, current or history of cancer, BMI, and left ventricular ejection fraction were associated with increased risk of MACE, and all predictors correlated significantly with MACE (abnormal concordance: HR 4.2, p = 0.043; current or history of cancer: HR 4.0, p = 0.047; BMI: HR 0.8, p = 0.020; left ventricular ejection fraction: HR 0.9, p = 0.017). Although these results support performing percutaneous coronary intervention according to evidence-based RFR or FFR thresholds, deferred lesions with discordant FFR and RFR results were not associated with worse prognosis.

Hva22, a REEP family protein in fission yeast, promotes reticulophagy in collaboration with a receptor protein.

The endoplasmic reticulum (ER) undergoes selective autophagy called reticulophagy or ER-phagy. Multiple reticulon- and receptor expression enhancing protein (REEP)-like ER-shaping proteins, including budding yeast Atg40, serve as reticulophagy receptors that stabilize the phagophore on the ER by interacting with phagophore-conjugated Atg8. Additionally, they facilitate phagophore engulfment of the ER by remodeling ER morphology. We reveal that Hva22, a REEP family protein in fission yeast, promotes reticulophagy without Atg8-binding capacity. The role of Hva22 in reticulophagy can be replaced by expressing Atg40 independently of its Atg8-binding ability. Conversely, adding an Atg8-binding sequence to Hva22 enables it to substitute for Atg40 in budding yeast. Thus, the phagophore-stabilizing and ER-shaping activities, both of which Atg40 solely contains, are divided between two separate factors, receptors and Hva22, respectively, in fission yeast.Abbreviations: AIM: Atg8-family interacting motif; Atg: autophagy related; DTT: dithiothreitol; ER: endoplasmic reticulum GFP: green fluorescent protein; NAA: 1-naphthaleneacetic acid; REEP: receptor expression enhancing protein; RFP: red fluorescent protein; UPR: unfolded protein response.

The mitochondrial intermembrane space protein mitofissin drives mitochondrial fission required for mitophagy.

Mitophagy plays an important role in mitochondrial homeostasis by selective degradation of mitochondria. During mitophagy, mitochondria should be fragmented to allow engulfment within autophagosomes, whose capacity is exceeded by the typical mitochondria mass. However, the known mitochondrial fission factors, dynamin-related proteins Dnm1 in yeasts and DNM1L/Drp1 in mammals, are dispensable for mitophagy. Here, we identify Atg44 as a mitochondrial fission factor that is essential for mitophagy in yeasts, and we therefore term Atg44 and its orthologous proteins mitofissin. In mitofissin-deficient cells, a part of the mitochondria is recognized by the mitophagy machinery as cargo but cannot be enwrapped by the autophagosome precursor, the phagophore, due to a lack of mitochondrial fission. Furthermore, we show that mitofissin directly binds to lipid membranes and brings about lipid membrane fragility to facilitate membrane fission. Taken together, we propose that mitofissin acts directly on lipid membranes to drive mitochondrial fission required for mitophagy.

Evaluation of the Safety and Effectiveness of Coil Embolization for Anterior Communicating and Anterior Cerebral Artery Aneurysms.

Objective: Endovascular coil embolization for anterior communicating artery (ACoA) and anterior cerebral artery (ACA) aneurysms is associated with high total and near-total occlusion rates, but the complication rate is high. The development of newer endovascular technologies may improve the clinical outcomes. This study investigated the status of endovascular treatment of ACoA and ACA aneurysms by comparing our results with past reports. Methods: Between January 2006 and December 2018, we investigated 50 patients who were followed for 12 months or longer to clarify the outcomes of coil embolization. The outcomes of embolization were evaluated using time-of-flight MRA. The safety was evaluated based on procedure-related complications that affected clinical outcomes. Results: Initial assessments demonstrated complete obliteration in 84% (42 of 50 patients) and a residual neck in 14% (7 of 50 patients). Procedure-related complications developed in 12% (6 of 50 patients). The procedure-related morbidity rate was 2% (1 of 50 patients) and there was no procedure-related death. Recanalization was noted in 14% (7 of 50 patients, median follow-up period, 57 months). The recanalized aneurysms were significantly smaller than the stable aneurysms in maximum size (4.3 mm vs. 5.8 mm; p = 0.017) and height (3.7 mm vs. 4.3 mm; p = 0.035). Conclusion: We demonstrated the safety and effectiveness of endovascular coil embolization for ACoA and ACA aneurysms. The small size of aneurysms may be related to recanalization.

Transarterial and Transvenous Coil Embolization of Direct Carotid-Cavernous Fistulas.

Objective: Transvenous embolization (TVE) is typically used in combination with the residual shunt of transarterial embolization (TAE) for the treatment of direct carotid-cavernous fistulas (direct CCFs). This report is about our additional embolization method using combination therapy. Case Presentation: Five consecutive cases of direct CCF were presented; two were caused by aneurysms and three by head injuries. The treatment for each was started with TAE, with the addition of TVE if a shunt remained. At the time of TVE, a microcatheter positioned in the internal carotid artery passing from the cavernous sinus through the aneurysm neck or fistula was pulled back (pull-back method). It was then placed in the coil mass with TAE, and additional coils were filled. In two cases, the shunt disappeared by using only TAE, whereas it disappeared after being additionally embolized by the pull-back method in the remaining cases. All patients recovered with no postoperative complications. Conclusion: The TAE and TVE combination therapy with the pull-back method could efficiently embolize the residual shunt after TAE.

Mitophagy reporter mouse analysis reveals increased mitophagy activity in disuse-induced muscle atrophy.

Muscle disuse induces atrophy through increased reactive oxygen species (ROS) released from damaged mitochondria. Mitophagy, the autophagic degradation of mitochondria, is associated with increased ROS production. However, the mitophagy activity status during disuse-induced muscle atrophy has been a subject of debate. Here, we developed a new mitophagy reporter mouse line to examine how disuse affected mitophagy activity in skeletal muscles. Mice expressing tandem mCherry-EGFP proteins on mitochondria were then used to monitor the dynamics of mitophagy activity. The reporter mice demonstrated enhanced mitophagy activity and increased ROS production in atrophic soleus muscles following a 14-day hindlimb immobilization. Results also showed an increased expression of multiple mitophagy genes, including Bnip3, Bnip3l, and Park2. Our findings thus conclude that disuse enhances mitophagy activity and ROS production in atrophic skeletal muscles and suggests that mitophagy is a potential therapeutic target for disuse-induced muscle atrophy.

Mitophagy regulation mediated by the Far complex in yeast.

Mitochondrial autophagy (mitophagy) selectively degrades mitochondria and plays an important role in mitochondrial homeostasis. In the yeast Saccharomyces cerevisiae, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy, whereas this phosphorylation is counteracted by the protein phosphatase Ppg1. Although Ppg1 functions cooperatively with the Far complex (Far3, Far7, Far8, Vps64/Far9, Far10 and Far11), their relationship and the underlying phosphoregulatory mechanism of Atg32 remain unclear. Our recent study revealed: (i) the Far complex plays its localization-dependent roles, regulation of mitophagy and target of rapamycin complex 2 (TORC2) signaling, via the mitochondria- and endoplasmic reticulum (ER)-localized Far complexes, respectively; (ii) Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required to assemble the sub- and core-Far complexes; (iii) association and dissociation between the Far complex and Atg32 are crucial determinants for mitophagy regulation. Here, we summarize our findings and discuss unsolved issues.

Downregulation of the ypdA Gene Encoding an Intermediate of His-Asp Phosphorelay Signaling in Aspergillus nidulans Induces the Same Cellular Effects as the Phenylpyrrole Fungicide Fludioxonil.

Many eukaryotic histidine-to-aspartate (His-Asp) phosphorelay systems consist of three types of signal transducers: a His-kinase (HK), a response regulator (RR), and a histidine-containing phosphotransfer intermediate (HPt). In general, the HPt acts as an intermediate between the HK and the RR and is indispensable for inducing appropriate responses to environmental stresses. In a previous study, we attempted but were unable to obtain deletion mutants of the ypdA gene in order to characterize its function in the filamentous fungus Aspergillus nidulans. In the present study, we constructed the CypdA strain in which ypdA expression is conditionally regulated by the A. nidulans alcA promoter. We constructed CypdA strains with RR gene disruptions (CypdA-sskAΔ, CypdA-srrAΔ, and CypdA-sskAΔsrrAΔ). Suppression of YpdA induced by ypdA downregulation activated the downstream HogA mitogen-activated protein kinase cascade. YpdA suppression caused severe growth defects and abnormal hyphae, with features such as enhanced septation, a decrease in number of nuclei, nuclear fragmentation, and hypertrophy of vacuoles, both regulated in an SskA-dependent manner. Fludioxonil treatment caused the same cellular responses as ypdA suppression. The growth-inhibitory effects of fludioxonil and the lethality caused by ypdA downregulation may be caused by the same or similar mechanisms and to be dependent on both the SskA and SrrA pathways.

Association and dissociation between the mitochondrial Far complex and Atg32 regulate mitophagy.

Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.

Atg43 tethers isolation membranes to mitochondria to promote starvation-induced mitophagy in fission yeast.

Degradation of mitochondria through mitophagy contributes to the maintenance of mitochondrial function. In this study, we identified that Atg43, a mitochondrial outer membrane protein, serves as a mitophagy receptor in the model organism Schizosaccharomyces pombe to promote the selective degradation of mitochondria. Atg43 contains an Atg8-family-interacting motif essential for mitophagy. Forced recruitment of Atg8 to mitochondria restores mitophagy in Atg43-deficient cells, suggesting that Atg43 tethers expanding isolation membranes to mitochondria. We found that the mitochondrial import factors, including the Mim1-Mim2 complex and Tom70, are crucial for mitophagy. Artificial mitochondrial loading of Atg43 bypasses the requirement of the import factors, suggesting that they contribute to mitophagy through Atg43. Atg43 not only maintains growth ability during starvation but also facilitates vegetative growth through its mitophagy-independent function. Thus, Atg43 is a useful model to study the mechanism and physiological roles, as well as the origin and evolution, of mitophagy in eukaryotes.

Limited cutaneous systemic sclerosis with gangrene: an autopsy case.

We describe an autopsy case of a 75-year-old female with limited cutaneous systemic sclerosis (lcSSc) and gangrene due to macrovascular involvement. She was diagnosed with lcSSc complicated with pulmonary arterial hypertension and digital ulcers 9 years before admission. She had recurrent and refractory lower limb ulcers (LLUs), and died because of sepsis caused by gangrene infection. Autopsy findings revealed severely thickened arterial walls of the visceral organs, consistent with vascular involvement of SSc. Systemic vascular involvement in lcSSc may progress in patients with LLUs who harbour several risk factors for vascular involvement.

Regulatory Mechanisms of Mitochondrial Autophagy: Lessons From Yeast.

Mitochondria produce the majority of ATP required by cells via oxidative phosphorylation. Therefore, regulation of mitochondrial quality and quantity is important for maintaining cellular activities. Mitophagy, the selective degradation of mitochondria, is thought to contribute to control of mitochondrial quality and quantity. In recent years, the molecular mechanism of mitophagy has been extensively studied in yeast and mammalian cells. In particular, identification of the mitophagy receptor Atg32 has contributed to substantial progress in understanding of mitophagy in yeast. This review summarizes the molecular mechanism of mitophagy in yeast and compares it to the mechanism of mitophagy in mammals. We also discuss the current understanding of mitophagy in plants.

Characteristics of Gut Microbiota in Patients With Diabetes Determined by Data Mining Analysis of Terminal Restriction Fragment Length Polymorphisms.

BACKGROUND: This study was performed to clarify whether gut microbiota obtained from fecal samples could identify the type of diabetes in patients of each gender by using a combination of terminal restriction fragment length polymorphism (T-RFLP) analysis and data mining. METHODS: A cross-sectional study was performed at three centers. Fecal samples were collected from 12 Japanese patients with type 1 diabetes mellitus (T1D), 18 patients with type 2 diabetes mellitus (T2D), and 31 subjects without diabetes mellitus (non-DM). Amplification of fecal 16S rRNA was carried out. After digestion of the amplification products with restriction enzymes (AluI, BslI, HaeIII, and MspI), terminal restriction fragments (T-RFs) of DNA were detected. A data mining algorithm (classification and regression tree (CART) modeling system) provides a decision tree that classifies subjects into various groups according to pre-assigned characteristics. RESULTS: Among men, the error rate was 2.4% with MspI, while error rates were 0.0% with other restriction enzymes. Among women, the error rate was 0.0% with all restriction enzymes. The operational taxonomic units (OTUs) incorporated into the decision tree differed between men and women. CONCLUSIONS: We were able to classify the 16SrRNA gene amplification products obtained from fecal samples of T1D patients, T2D patients, and non-DM subjects with a high level of precision by combining T-RFLP analysis and data mining. Specific gut microbiota patterns were found for T1D and T2D patients, as well as a sex difference of the patterns.

PP2A-like protein phosphatase Ppg1: an emerging negative regulator of mitophagy in yeast.

Macroautophagy/autophagy (bulk autophagy) is a catabolic process that nonselectively degrades cytoplasmic proteins and organelles. In contrast to bulk autophagy, selective types of autophagy target specific cellular components as cargos, whereas their specific receptor proteins play central roles in cargo selection. In the yeast Saccharomyces cerevisiae, receptor proteins for the cytoplasm-to-vacuole targeting pathway, mitophagy, and pexophagy are phosphoregulated by kinases. This phosphorylation facilitates interaction with the scaffold/adaptor protein Atg11, subsequently recruiting core autophagy proteins to initiate autophagosome formation. However, the molecular mechanism inhibiting this phosphorylation to prevent unrequired selective autophagy remains unknown. Our recent study revealed that the protein phosphatase 2A-like protein phosphatase Ppg1 and its associated Far complex cooperatively inhibit mitophagy by counteracting casein kinase 2-mediated phosphorylation of the mitophagy receptor Atg32. Herein, we summarize our findings regarding Ppg1 and pose unanswered questions.

The PP2A-like Protein Phosphatase Ppg1 and the Far Complex Cooperatively Counteract CK2-Mediated Phosphorylation of Atg32 to Inhibit Mitophagy.

Mitophagy plays an important role in mitochondrial quality control. In yeast, phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 (CK2) upon induction of mitophagy is a prerequisite for interaction of Atg32 with Atg11 (an adaptor protein for selective autophagy) and following delivery of mitochondria to the vacuole for degradation. Because CK2 is constitutively active, Atg32 phosphorylation must be precisely regulated to prevent unrequired mitophagy. We found that the PP2A (protein phosphatase 2A)-like protein phosphatase Ppg1 was essential for dephosphorylation of Atg32 and inhibited mitophagy. We identified the Far complex proteins, Far3, Far7, Far8, Far9, Far10, and Far11, as Ppg1-binding proteins. Deletion of Ppg1 or Far proteins accelerated mitophagy. Deletion of a cytoplasmic region (amino acid residues 151-200) of Atg32 caused the same phenotypes as in ppg1Δ cells, which suggested that dephosphorylation of Atg32 by Ppg1 required this region. Therefore, Ppg1 and the Far complex cooperatively dephosphorylate Atg32 to prevent excessive mitophagy.