Peroxisome-Mediated Metabolism Is Required for Immune Response to Microbial Infection.

The innate immune response is critical for animal homeostasis and is conserved from invertebrates to vertebrates. This response depends on specialized cells that recognize, internalize, and destroy microbial invaders through phagocytosis. This is coupled to autonomous or non-autonomous cellular signaling via reactive oxygen species (ROS) and cytokine production. Lipids are known signaling factors in this process, as the acute phase response of macrophages is accompanied by systemic lipid changes that help resolve inflammation. We found that peroxisomes, membrane-enclosed organelles central to lipid metabolism and ROS turnover, were necessary for the engulfment of bacteria by Drosophila and mouse macrophages. Peroxisomes were also required for resolution of bacterial infection through canonical innate immune signaling. Reduced peroxisome function impaired the turnover of the oxidative burst necessary to fight infection. This impaired response to bacterial challenge affected cell and organism survival and revealed a previously unknown requirement for peroxisomes in phagocytosis and innate immunity.

Thioredoxin-interacting protein is essential for memory T cell formation via the regulation of the redox metabolism.

CD4+ memory T cells are central to long-lasting protective immunity and are involved in shaping the pathophysiology of chronic inflammation. While metabolic reprogramming is critical for the generation of memory T cells, the mechanisms controlling the redox metabolism in memory T cell formation remain unclear. We found that reactive oxygen species (ROS) metabolism changed dramatically in T helper-2 (Th2) cells during the contraction phase in the process of memory T cell formation. Thioredoxin-interacting protein (Txnip), a regulator of oxidoreductase, regulated apoptosis by scavenging ROS via the nuclear factor erythroid 2-related factor 2 (Nrf2)-biliverdin reductase B (Blvrb) pathway. Txnip regulated the pathology of chronic airway inflammation in the lung by controlling the generation of allergen-specific pathogenic memory Th2 cells in vivo. Thus, the Txnip-Nrf2-Blvrb axis directs ROS metabolic reprogramming in Th2 cells and is a potential therapeutic target for intractable chronic inflammatory diseases.

Singlet oxygen-mediated photochemical cross-linking of an engineered fluorescent flavoprotein iLOV.

Genetically encoded photoactive proteins are integral tools in modern biochemical and molecular biological research. Within this tool box, truncated variants of the phototropin two light-oxygen-voltage flavoprotein have been developed to photochemically generate singlet oxygen (1O2) in vitro and in vivo, yet the effect of 1O2 on these genetically encoded photosensitizers remains underexplored. In this study, we demonstrate that the "improved" light-oxygen-voltage flavoprotein is capable of photochemical 1O2 generation. Once generated, 1O2 induces protein oligomerization via covalent cross-linking. The molecular targets of protein oligomerization by cross-linking are not endogenous tryptophans or tyrosines, but rather primarily histidines. Substitution of surface-exposed histidines for serine or glycine residues effectively eliminates protein cross-linking. When used in biochemical applications, such protein-protein cross-links may interfere with native biological responses to 1O2, which can be ameliorated by substitution of the surface exposed histidines of improved" light-oxygen-voltage or other 1O2-generating flavoproteins.

N-acetyl cysteine turns EPAC activators into potent killers of acute lymphoblastic leukemia cells.

Today, the majority of patients with pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL, hereafter ALL) survive their disease, but many of the survivors suffer from life-limiting late effects of the treatment. ALL develops in the bone marrow, where the cells are exposed to cAMP-generating prostaglandin E2. We have previously identified the cAMP signaling pathway as a putative target for improved efficacy of ALL treatment, based on the ability of cAMP signaling to reduce apoptosis induced by DNA damaging agents. In the present study, we have identified the antioxidant N-acetyl cysteine (NAC) as a powerful modifier of critical events downstream of the cell-permeable cAMP analog 8-(4-chlorophenylthio) adenosine-3', 5'- cyclic monophosphate (8-CPT). Accordingly, we found NAC to turn 8-CPT into a potent killer of ALL cells in vitro both in the presence and absence of DNA damaging treatment. Furthermore, we revealed that NAC in combination with 8-CPT is able to delay the progression of ALL in a xenograft model in NOD-scid IL2Rγnull mice. NAC was shown to rely on the ability of 8-CPT to activate the guanine-nucleotide exchange factor EPAC, and we demonstrated that the ALL cells are killed by apoptosis involving sustained elevated levels of calcium imposed by the combination of the two drugs. Taken together, we propose that 8-CPT in the presence of NAC might be utilized as a novel strategy for treating pediatric ALL patients, and that this powerful combination might be exploited to enhance the therapeutic index of current ALL targeting therapies.

Development of a mouse model expressing a bifunctional glutathione-synthesizing enzyme to study glutathione limitation in vivo.

Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.

FDX2, an iron-sulfur cluster assembly factor, is essential to prevent cellular senescence, apoptosis or ferroptosis of ovarian cancer cells.

Recent studies reveal that biosynthesis of iron-sulfur clusters (Fe-Ss) is essential for cell proliferation, including that of cancer cells. Nonetheless, it remains unclear how Fe-S biosynthesis functions in cell proliferation/survival. Here, we report that proper Fe-S biosynthesis is essential to prevent cellular senescence, apoptosis, or ferroptosis, depending on cell context. To assess these outcomes in cancer, we developed an ovarian cancer line with conditional KO of FDX2, a component of the core Fe-S assembly complex. FDX2 loss induced global downregulation of Fe-S-containing proteins and Fe2+ overload, resulting in DNA damage and p53 pathway activation, and driving the senescence program. p53 deficiency augmented DNA damage responses upon FDX2 loss, resulting in apoptosis rather than senescence. FDX2 loss also sensitized cells to ferroptosis, as evidenced by compromised redox homeostasis of membrane phospholipids. Our results suggest that p53 status and phospholipid homeostatic activity are critical determinants of diverse biological outcomes of Fe-S deficiency in cancer cells.

A glutathione S-transferase regulates lignin biosynthesis and enhances salt tolerance in tomato.

Salt stress adversely affects the growth and yield of crops. Glutathione S-transferases (GSTs) are involved in plant growth and responses to biotic and abiotic stresses. In this study, 400 mM NaCl stress significantly induced the expression of Glutathione S-transferase U43 (SlGSTU43) in the roots of the wild-type tomato (Solanum lycopersicum L.) plants. Overexpressing SlGSTU43 enhanced the ability of scavenging reactive oxygen species (ROS) in tomato leaves and roots under NaCl stress, while SlGSTU43 knock-out mutants showed the opposite performance. RNA sequencing analysis revealed that overexpressing SlGSTU43 affected the expression of genes related to lignin biosynthesis. We demonstrated that SlGSTU43 can regulate the lignin content in tomato through its interaction with SlCOMT2, a key enzyme involved in lignin biosynthesis, and promote the growth of tomato plants under NaCl stress. In addition, SlMYB71 and SlWRKY8 interact each other, and can directly bind to the promoter of SlGSTU43 to transcriptionally activate its expression separately or in combination. When SlMYB71 and SlWRKY8 were silenced in tomato plants individually or collectively, the plants were sensitive to NaCl stress, and their GST activities and lignin contents decreased. Our research indicates that SlGSTU43 can enhance salt stress tolerance in tomato by regulating lignin biosynthesis, which is regulated by interacting with SlCOMT2, as well as SlMYB71 and SlWRKY8. This finding broadens our understanding of GST functions.

A broadly applicable, stress-mediated bacterial death pathway regulated by the phosphotransferase system (PTS) and the cAMP-Crp cascade.

Recent work indicates that killing of bacteria by diverse antimicrobial classes can involve reactive oxygen species (ROS), as if a common, self-destructive response to antibiotics occurs. However, the ROS-bacterial death theory has been challenged. To better understand stress-mediated bacterial death, we enriched spontaneous antideath mutants of Escherichia coli that survive treatment by diverse bactericidal agents that include antibiotics, disinfectants, and environmental stressors, without a priori consideration of ROS. The mutants retained bacteriostatic susceptibility, thereby ruling out resistance. Surprisingly, pan-tolerance arose from carbohydrate metabolism deficiencies in ptsI (phosphotransferase) and cyaA (adenyl cyclase); these genes displayed the activity of upstream regulators of a widely shared, stress-mediated death pathway. The antideath effect was reversed by genetic complementation, exogenous cAMP, or a Crp variant that bypasses cAMP binding for activation. Downstream events comprised a metabolic shift from the TCA cycle to glycolysis and to the pentose phosphate pathway, suppression of stress-mediated ATP surges, and reduced accumulation of ROS. These observations reveal how upstream signals from diverse stress-mediated lesions stimulate shared, late-stage, ROS-mediated events. Cultures of these stable, pan-tolerant mutants grew normally and were therefore distinct from tolerance derived from growth defects described previously. Pan-tolerance raises the potential for unrestricted disinfectant use to contribute to antibiotic tolerance and resistance. It also weakens host defenses, because three agents (hypochlorite, hydrogen peroxide, and low pH) affected by pan-tolerance are used by the immune system to fight infections. Understanding and manipulating the PtsI-CyaA-Crp­mediated death process can help better control pathogens and maintain beneficial microbiota during antimicrobial treatment.

Reactive Oxygen Species Responsive Supramolecular Prodrug Eyedrops for the Treatment of Choroidal Neovascularization.

Choroidal neovascularization (CNV) represents a hallmark of neovascular fundus diseases, including age-related macular degeneration and diabetic retinopathy. Traditional eyedrops have encountered formidable challenges in treating CNV, primarily due to their extremely poor intraocular bioavailability and potential adverse off-target effects. Herein, an ocular-permeable supramolecular prodrug eyedrop (Di-DAS/P-PCD) has been developed for the on-demand delivery of antiangiogenic agents in the oxidative microenvironment of CNV. The eyedrop nanoformulation is composed of cell-penetrating peptide-modified PEGylated cyclodextrin (P-PCD) and reactive oxygen species (ROS)-sensitive antiangiogenic dasatinib prodrug Di-DAS. In a laser-induced CNV mouse model, daily instillation of Di-DAS/P-PCD has achieved remarkable penetration into the choroid and significantly suppressed CNV growth while exhibiting a good biocompatibility profile. Our results highlight the potential of the supramolecular prodrug eyedrops as a versatile approach for the targeted treatment of CNV and other neovascular eye disorders.

The Synergy between Antibiotics and the Nanoparticle-Based Photodynamic Effect.

Antimicrobial resistance (AMR) is a growing global health concern, necessitating innovative strategies beyond the development of new antibiotics. Here, we employed NdYVO4:Eu3+ nanoparticles, which can persistently produce reactive oxygen species (ROS) after stopping the light, as a model of photodynamic nanoparticles and demonstrated that the photodynamic effect can serve as an adjuvant with antibiotics to effectively reduce their minimum inhibitory concentration. These preirradiated nanoparticles could penetrate the bacterial cell membrane, significantly enhancing the potency of antibiotics. We showed that the synergy effect could be attributed to disrupting crucial cellular processes by ROS, including damaging cell membrane proteins, interfering with energy supply, and inhibiting antibiotic metabolism. Our findings suggested that complementing the photodynamic effect might be a robust strategy to enhance antibiotic potency, providing an alternative antibacterial treatment paradigm.

Porous Semiconducting Polymer Nanoparticles as Intracellular Biophotonic Mediators to Modulate the Reactive Oxygen Species Balance.

The integration of nanotechnology with photoredox medicine has led to the emergence of biocompatible semiconducting polymer nanoparticles (SPNs) for the optical modulation of intracellular reactive oxygen species (ROS). However, the need for efficient photoactive materials capable of finely controlling the intracellular redox status with high spatial resolution at a nontoxic light density is still largely unmet. Herein, highly photoelectrochemically efficient photoactive polymer beads are developed. The photoactive material/electrolyte interfacial area is maximized by designing porous semiconducting polymer nanoparticles (PSPNs). PSPNs are synthesized by selective hydrolysis of the polyester segments of nanoparticles made of poly(3-hexylthiophene)-graft-poly(lactic acid) (P3HT-g-PLA). The photocurrent of PSPNs is 4.5-fold higher than that of nonporous P3HT-g-PLA-SPNs, and PSPNs efficiently reduce oxygen in an aqueous environment. PSPNs are internalized within endothelial cells and optically trigger ROS generation with a >1.3-fold concentration increase with regard to nonporous P3HT-SPNs, at a light density as low as a few milliwatts per square centimeter, fully compatible with in vivo, chronic applications.

Antioxidant peptides, the guardian of life from oxidative stress.

Reactive oxygen species (ROS) are produced during oxidative metabolism in aerobic organisms. Under normal conditions, ROS production and elimination are in a relatively balanced state. However, under internal or external environmental stress, such as high glucose levels or UV radiation, ROS production can increase significantly, leading to oxidative stress. Excess ROS production not only damages biomolecules but is also closely associated with the pathogenesis of many diseases, such as skin photoaging, diabetes, and cancer. Antioxidant peptides (AOPs) are naturally occurring or artificially designed peptides that can reduce the levels of ROS and other pro-oxidants, thus showing great potential in the treatment of oxidative stress-related diseases. In this review, we discussed ROS production and its role in inducing oxidative stress-related diseases in humans. Additionally, we discussed the sources, mechanism of action, and evaluation methods of AOPs and provided directions for future studies on AOPs.

The interplay of DNA damage and repair, gene expression, and mutagenesis in mammalian cells during oxidative stress.

We investigated the interplay among oxidative DNA damage and repair, expression of genes encoding major base excision repair (BER) enzymes and bypass DNA polymerases, and mutagenesis in mammalian cells. Primary mouse embryonic fibroblasts were challenged with oxidative stress induced by methylene blue plus visible light, and formation and repair of DNA damage, changes in gene expression, and mutagenesis were determined at increasing intervals post-treatment (0 - 192 hours). Significant formation of oxidative DNA damage together with upregulation of Ogg1, Polß, and Polκ, and no changes in Mutyh and Nudt1 expression were found in treated cells. There was a distinct interconnection between Ogg1 and Polß expression and DNA damage formation and repair whereby changes in expression of these two genes were proportionate to the levels of oxidative DNA damage, once a 3-plus hour lag time passed (P < 0.05). Equally notable was the matching pattern of Polκ expression and kinetics of oxidative DNA damage and repair (P < 0.05). The DNA damage and gene expression data were remarkably consistent with mutagenicity data in the treated cells; the induced mutation spectrum is indicative of erroneous bypass of oxidized DNA bases and incorporation of oxidized deoxynucleoside triphosphates during replication of the genomic DNA. Our findings support follow-up functional studies to elucidate how oxidation of DNA bases and the nucleotide pool, overexpression of Polκ, delayed upregulation of Ogg1 and Polß, and inadequate expression of Nudt1 and Mutyh collectively affect mutagenesis consequent to oxidative stress.

Role of oxidative stress in the concurrent development of osteoporosis and tendinopathy: Emerging challenges and prospects for treatment modalities.

Both osteoporosis and tendinopathy are widely prevalent disorders, encountered in diverse medical contexts. Whilst each condition has distinct pathophysiological characteristics, they share several risk factors and underlying causes. Notably, oxidative stress emerges as a crucial intersecting factor, playing a pivotal role in the onset and progression of both diseases. This imbalance arises from a dysregulation in generating and neutralising reactive oxygen species (ROS), leading to an abnormal oxidative environment. Elevated levels of ROS can induce multiple cellular disruptions, such as cytotoxicity, apoptosis activation and reduced cell function, contributing to tissue deterioration and weakening the structural integrity of bones and tendons. Antioxidants are substances that can prevent or slow down the oxidation process, including Vitamin C, melatonin, resveratrol, anthocyanins and so on, demonstrating potential in treating these overlapping disorders. This comprehensive review aims to elucidate the complex role of oxidative stress within the interlinked pathways of these comorbid conditions. By integrating contemporary research and empirical findings, our objective is to outline new conceptual models and innovative treatment strategies for effectively managing these prevalent diseases. This review underscores the importance of further in-depth research to validate the efficacy of antioxidants and traditional Chinese medicine in treatment plans, as well as to explore targeted interventions focused on oxidative stress as promising areas for future medical advancements.

TRPM2 channels are essential for regulation of cytokine production in lung interstitial macrophages.

Interstitial macrophages (IMs) are essential for organ homeostasis, inflammation, and autonomous immune response in lung tissues, which are achieved through polarization to a pro-inflammatory M1 and an M2 state for tissue repair. Their remote parenchymal localization and low counts, however, are limiting factors for their isolation and molecular characterization of their specific role during tissue inflammation. We isolated viable murine IMs in sufficient quantities by coculturing them with stromal cells and analyzed mRNA expression patterns of transient receptor potential (TRP) channels in naïve and M1 polarized IMs after application of lipopolysaccharide (LPS) and interferon γ. M-RNAs for the second member of the melastatin family of TRP channels, TRPM2, were upregulated in the M1 state and functional channels were identified by their characteristic currents induced by ADP-ribose, its specific activator. Most interestingly, cytokine production and secretion of interleukin-1α (IL-1α), IL-6 and tumor necrosis factor-α in M1 polarized but TRPM2-deficient IMs was significantly enhanced compared to WT cells. Activation of TRPM2 channels by ADP-ribose (ADPR) released from mitochondria by ROS-produced H2O2 significantly increases plasma membrane depolarization, which inhibits production of reactive oxygen species by NADPH oxidases and reduces cytokine production and secretion in a negative feedback loop. Therefore, TRPM2 channels are essential for the regulation of cytokine production in M1-polarized murine IMs. Specific activation of these channels may promote an anti-inflammatory phenotype and prevent a harmful cytokine storm often observed in COVID-19 patients.

Alternative magnetic field exposure suppresses tumor growth via metabolic reprogramming.

Application of physical forces, ranging from ultrasound to electric fields, is recommended in various clinical practice guidelines, including those for treating cancers and bone fractures. However, the mechanistic details of such treatments are often inadequately understood, primarily due to the absence of comprehensive study models. In this study, we demonstrate that an alternating magnetic field (AMF) inherently possesses a direct anti-cancer effect by enhancing oxidative phosphorylation (OXPHOS) and thereby inducing metabolic reprogramming. We observed that the proliferation of human glioblastoma multiforme (GBM) cells (U87 and LN229) was inhibited upon exposure to AMF within a specific narrow frequency range, including around 227 kHz. In contrast, this exposure did not affect normal human astrocytes (NHA). Additionally, in mouse models implanted with human GBM cells in the brain, daily exposure to AMF for 30 min over 21 days significantly suppressed tumor growth and prolonged overall survival. This effect was associated with heightened reactive oxygen species (ROS) production and increased manganese superoxide dismutase (MnSOD) expression. The anti-cancer efficacy of AMF was diminished by either a mitochondrial complex IV inhibitor or a ROS scavenger. Along with these observations, there was a decrease in the extracellular acidification rate (ECAR) and an increase in the oxygen consumption rate (OCR). This suggests that AMF-induced metabolic reprogramming occurs in GBM cells but not in normal cells. Our results suggest that AMF exposure may offer a straightforward strategy to inhibit cancer cell growth by leveraging oxidative stress through metabolic reprogramming.

Inhibition of insulin degrading enzyme suppresses osteoclast hyperactivity via enhancing Nrf2-dependent antioxidant response in glucocorticoid-induced osteonecrosis of the femoral head.

BACKGROUND: Osteoclast hyperactivation due to the pathological overproduction of reactive oxygen species (ROS) stimulated by glucocorticoids (GCs) is one of the key drivers behind glucocorticoid-induced osteonecrosis of the femoral head (GIONFH). The insulin degrading enzyme (IDE), a conserved Zn2+ metallo-endopeptidase, facilitates the DNA binding of glucocorticoid receptor and plays a substantial role in steroid hormone-related signaling pathways. However, the potential role of IDE in the pathogenesis of GIONFH is yet undefined. METHODS: In this study, we employed network pharmacology and bioinformatics analysis to explore the impact of IDE inhibition on GIONFH with 6bK as an inhibitory agent. Further evidence was collected through in vitro osteoclastogenesis experiments and in vivo evaluations involving methylprednisolone (MPS)-induced GIONFH mouse model. RESULTS: Enrichment analysis indicated a potential role of 6bK in redox regulation amid GIONFH development. In vitro findings revealed that 6bK could attenuate GCs-stimulated overactivation of osteoclast differentiation by interfering with the transcription and expression of key osteoclastic genes (Traf6, Nfatc1, and Ctsk). The use of an H2DCFDA probe and subsequent WB assays introduced the inhibitory effects of 6bK on osteoclastogenesis, linked with the activation of the nuclear factor erythroid-derived 2-like 2 (Nrf2)-mediated antioxidant system. Furthermore, Micro-CT scans validated that 6bK could alleviate GIONFH in MPS-induced mouse models. CONCLUSIONS: Our findings suggest that 6bK suppresses osteoclast hyperactivity in GCs-rich environment. This is achieved by reducing the accumulation of intracellular ROS via promoting the Nrf2-mediated antioxidant system, thus implying that IDE could be a promising therapeutic target for GIONFH.

Exploring the therapeutic potential of isoorientin in the treatment of osteoporosis: a study using network pharmacology and experimental validation.

BACKGROUND: Isoorientin (ISO) is a glycosylated flavonoid with antitumor, anti-inflammatory, and antioxidant properties. However, its effects on bone metabolism remain largely unknown. METHODS: In this study, we aimed to investigate the effects of ISO on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation in vitro and bone loss in post-ovariectomy (OVX) rats, as well as to elucidate the underlying mechanism. First, network pharmacology analysis indicated that MAPK1 and AKT1 may be potential therapeutic targets of ISO and that ISO has potential regulatory effects on the mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathways, as well as oxidative stress. ISO was added to RAW264.7 cells stimulated by RANKL, and its effects on osteoclast differentiation were evaluated using tartrate-resistant acid phosphatase (TRAP) staining, TRAP activity measurement, and F-actin ring analysis. Reactive oxygen species (ROS) production in osteoclasts was detected using a ROS assay kit. The effects of ISO on RANKL-triggered molecular cascade response were further investigated by Western blotting, quantitative real-time polymerase chain reaction, and immunofluorescence staining. In addition, the therapeutic effects of ISO were evaluated in vivo. RESULTS: ISO inhibited osteoclastogenesis in a time- and concentration-dependent manner. Mechanistically, ISO downregulated the expression of the main transcription factor for osteoclast differentiation by inhibiting MAPK and PI3K/AKT1 signaling pathways. Moreover, ISO exhibited protective effects in OVX-induced bone loss rats. This was consistent with the results derived from network pharmacology. CONCLUSION: Our findings suggest a potential therapeutic utility of ISO in the management of osteoclast-associated bone diseases, including osteoporosis.

The role of alternative autophagy in cell viability and response to paclitaxel treatment in v-Ha-ras-transformed NIH 3T3 cells.

In confluent v-Ha-ras-transformed NIH 3T3 fibroblasts (Ras-NIH 3T3), LC3 downregulation may precede a decrease in canonical autophagy, thus contributing to cell survival. Herein, we aimed to investigate the role of alternative autophagy in the viability of long-term cultures of Ras-NIH 3T3 cells and their parental NIH 3T3 cells. As cell confluence increased with the culture period, the level of alternative autophagy, as assessed through Lamp2-Rab9 co-localization, gradually decreased in both cell lines. However, Ras-NIH 3T3 cells maintained higher levels of alternative autophagy than the parental cells did. Rab9 knockdown minimally affected NIH 3T3 cells while drastically reducing the viability of Ras-NIH 3T3 cells, which suggested that alternative autophagy plays a critical role in Ras-NIH 3T3 cells. In contrast, reactive oxygen species (ROS) production in Ras-NIH 3T3 cells was higher than that in NIH 3T3 cells during long-term culture. Moreover, NIH 3T3 cells exhibited a continual decrease in mitochondrial mass, whereas Ras-NIH 3T3 cells maintained high mitochondrial mass. Immunofluorescence analysis of mitochondrial membrane marker proteins and mitochondrial membrane potential (MMP) also demonstrated a temporal pattern of changes similar to those of mitochondrial mass. This finding could be attributed to the relatively higher level of alternative autophagy in Ras-NIH 3T3 cells facilitating the removal of damaged mitochondria. Paclitaxel treatment in Ras-NIH 3T3 cells induced an increase in canonical autophagy rates along with suppression of alternative autophagy. Ras-NIH 3T3 cells showed high sensitivity to paclitaxel at the early stage of culture, but as cell confluence increased, resistance to paclitaxel increased, showing a similar level of cell viability to the vehicle control group. The study findings suggest that alternative autophagy is more important than canonical autophagy for maintaining cell survival in response to an unfavorable environment, such as during high cell confluence and exposure to anticancer agents.

53BP1 regulates the self-renewal ability of neural stem/progenitor cells through modulating mitochondrial homeostasis.

The regulation of intracellular reactive oxygen species (ROS) levels is important for maintaining the self-renewal ability of neural stem/progenitor cells (NSCs). In this study, we demonstrate that 53BP1, a DNA damage response factor known to facilitate the repair of DNA double-strand breaks, supports the maintenance of NSC stemness. ReNcell VM human NSCs with depleted 53BP1 exhibited reduced self-renewal ability compared with control NSCs, as revealed by a decrease in neurosphere size and an increase in differentiation into neural or glial cells within an NSC culture. Furthermore, 53BP1 depletion elevated cellular ROS levels, accompanied by mitochondrial abnormalities. The reduced self-renewal ability and elevated ROS levels in 53BP1-deficient NSCs were restored with the treatment of a radical scavenger, N-acetyl-l-cysteine. In addition, we investigated the functional relationship in the NSC self-renewal ability between 53BP1 and ataxia-telangiectasia mutated (ATM) or forkhead box O3a (FOXO3a), factors required for mitochondrial homeostasis, and the maintenance of NSC stemness. We found that ATM inhibition or FOXO3a deficiency, in addition to 53BP1 deficiency, did not induce further NSC stemness impairment. Collectively, our findings show that 53BP1, by cooperatively functioning with ATM and FOXO3a, supports the maintenance of NSC stemness by modulating mitochondrial homeostasis.