UVB radiation exposure modulates mitophagy in embryonic cells of freshwater prawn Macrobrachium olfersii: Exploring a protective organelle quality control mechanism.

Aquatic environments are subject to ultraviolet B (UVB) radiation incidence, and its effects on organisms are dose-dependent. Besides DNA, mitochondria are an important target of this radiation that causes structural damage and impairs its functional dynamics. Here, we hypothesize that mitophagy acts as an organelle quality control mechanism to mitigate UVB impacts in embryonic cells. Then, freshwater prawn Macrobrachium olfersii embryos was used as a model to investigate the effects of UVB on genes (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) and proteins (TOM20, PINK1, p62 and LC3B) involved in mitophagy modulation. The choice of genes and proteins was based on the identification of mitochondrial membrane (Tomm20, Opa1 and TOM20), mediation of mitophagy (Pink1, Prkn and PINK1), and recognition of mitochondria by the autophagosome membrane (Sqstm1, Map1lc3, p62 and LC3B). First, the phylogeny of all genes presented bootstrap values >80 and conserved domains among crustacean species. Gene expression was inherently modulated during development, with transcripts (Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3) overexpressed in the initial and final stages of development. Moreover, UVB radiation induced upregulation of Tomm20, Opa1, Pink, Prkn, Sqstm1, and Map1lc3 genes at 6 h after exposure. Interestingly, after 12 h, the protein content of PINK1, p62, and LC3B increased, while TOM20 was not responsive. Despite UVB radiation's harmful effects on embryonic cells, the chronology of gene expression and protein content indicates rapid activation of mitophagy, serving as an organelle quality control mechanism, given the analyzed cells' integrity.

Elimination of damaged mitochondria during UVB-induced senescence is orchestrated by NIX-dependent mitophagy.

Skin aging is the result of two types of aging, "intrinsic aging" an inevitable consequence of physiologic and genetically determined changes and "extrinsic aging," which is dependent on external factors such as exposure to sunlight, smoking, and dietary habits. UVB causes skin injury through the generation of free radicals and other oxidative byproducts, also contributing to DNA damage. Appearance and accumulation of senescent cells in the skin are considered one of the hallmarks of aging in this tissue. Mitochondria play an important role for the development of cellular senescence, in particular stress-induced senescence of human cells. However, many aspects of mitochondrial physiology relevant to cellular senescence and extrinsic skin aging remain to be unraveled. Here, we demonstrate that mitochondria damaged by UVB irradiation of human dermal fibroblasts (HDF) are eliminated by NIX-dependent mitophagy and that this process is important for cell survival under these conditions. Additionally, UVB-irradiation of human dermal fibroblasts (HDF) induces the shedding of extracellular vesicles (EVs), and this process is significantly enhanced in UVB-irradiated NIX-depleted cells. Our findings establish NIX as the main mitophagy receptor in the process of UVB-induced senescence and suggest the release of EVs as an alternative mechanism of mitochondrial quality control in HDF.

Innate immune receptor NOD2 mediates LGR5+ intestinal stem cell protection against ROS cytotoxicity via mitophagy stimulation.

The nucleotide-binding oligomerization domain-containing protein 2 (NOD2) agonist muramyl dipeptide (MDP), a peptidoglycan motif common to all bacteria, supports leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)+ intestinal stem cell (ISC) survival through NOD2 activation upon an otherwise lethal oxidative stress-mediated signal. However, the underlying protective mechanisms remain unknown. Here, using irradiation as stressor and primarily murine-derived intestinal organoids as a model system, we show that MDP induced a significant reduction of total and mitochondrial reactive oxygen species (ROS) within ISCs, which was associated with mitophagy induction. ATG16L1 knockout (KO) and NOD2 KO organoids did not benefit from the MDP-induced cytoprotection. We confirmed the MDP-dependent induction of ISC mitophagy upon stress in vivo. These findings elucidate the NOD2-mediated mechanism of cytoprotection involving the clearance of the lethal excess of ROS molecules through mitophagy, triggered by the coordinated activation of NOD2 and ATG16L1 by a nuclear factor κB (NF-κB)-independent pathway.

AIE-Based Theranostic Agent: In Situ Tracking Mitophagy Prior to Late Apoptosis To Guide the Photodynamic Therapy.

Photodynamic therapy (PDT) takes advantage of reactive oxygen species (ROS) to trigger the apoptosis for cancer therapy. Given that cell apoptosis is a form of programmed cell death involved with multiple suborganelles and cancer cells are more sensitive to ROS than normal cells, early confirmation of the apoptosis induced by ROS would effectively avoid overtreatment. Herein, we highlight an aggregation-induced emission (AIE)-based theranostic agent (TPA3) to in situ dynamically track mitophagy prior to late apoptosis. TPA3 showed high specificity to autophagy vacuoles (AVs), of which appearance is the signature event of mitophagy during early apoptosis and delivered photocytotoxicity to cancer cells and skin cancer tumors in nude mice under irradiation of white light. Furthermore, in situ monitoring of the dynamical mitophagy process involved with mitochondria, AVs, and lysosomes was performed for the first time under confocal microscopy, providing a real-time self-monitoring system for assessing the curative effect prior to late apoptosis. This fluorescence imaging guided PDT witness great advances for applying in the clinical application.

Neuroprotective effect of anodal transcranial direct current stimulation on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice through modulating mitochondrial dynamics.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of protein inclusions and the loss of dopaminergic neurons. Abnormal mitochondrial homeostasis is thought to be important for the pathogenesis of PD. Transcranial direct current stimulation (tDCS), a noninvasive brain stimulation technique, constitutes a promising approach for promoting recovery of various neurological conditions. However, little is known about its mechanism of action. The present study elucidated the neuroprotective effects of tDCS on the mitochondrial quality control pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We used the MPTP-induced neurotoxicity in vivo model. Mice were stimulated for 5 consecutive days with MPTP treatment. After observation of behavioral alteration using the rotarod test, mice were sacrificed for the measurement of the PD- and mitochondrial quality control-related protein levels in the substantia nigra. tDCS improved the behavioral alterations and changes in tyrosine hydroxylase levels in MPTP-treated mice. Furthermore, tDCS attenuated mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in the MPTP-induced PD mouse model. MPTP significantly increased mitophagy and decreased mitochondrial biogenesis-related proteins. These changes were attenuated by tDCS. Furthermore, MPTP significantly increased fission-related protein dynamin-related protein 1 with no effect on fusion-related protein mitofusin-2, and tDCS attenuated these changes. Our findings demonstrated the neuroprotective effect of anodal tDCS on the MPTP-induced neurotoxic mouse model through suppressing excessive mitophagy and balancing mitochondrial dynamics. The neuroprotective effect of anodal tDCS with modulation of mitochondrial dynamics provides a new therapeutic strategy for the treatment of PD.

Sonodynamic therapy inhibits palmitate-induced beta cell dysfunction via PINK1/Parkin-dependent mitophagy.

In type 2 diabetes mellitus (T2DM), the overload of glucose and lipids can promote oxidative stress and inflammatory responses and contribute to the failure of beta cells. However, therapies that can modulate the function of beta cells and thus prevent their failure have not been well explored. In this study, beta cell injury model was established with palmitic acid (PA) to simulate the lipotoxicity (high-fat diet) found in T2DM. Sonodynamic therapy (SDT), a novel physicochemical treatment, was applied to treat injured beta cells. We found that SDT had specific effects on mitochondria and induced transient large amount of mitochondrial reactive oxygen species (ROS) production in beta cells. SDT also improved the morphology and function of abnormal mitochondria, inhibited inflammatory response and reduced beta cell dysfunction. The improvement of mitochondria was mediated by PINK1/Parkin-dependent mitophagy. Additionally, SDT rescued the transcription of PINK1 mRNA which was blocked by PA treatment, thus providing abundant PINK1 for mitophagy. Moreover, SDT also increased insulin secretion from beta cells. The protective effects of SDT were abrogated when mitophagy was inhibited by cyclosporin A (CsA). In summary, SDT potently inhibits lipotoxicity-induced beta cell failure via PINK1/Parkin-dependent mitophagy, providing theoretical guidance for T2DM treatment in aspects of islet protection.

UVB-induced inactivation of manganese-containing superoxide dismutase promotes mitophagy via ROS-mediated mTORC2 pathway activation.

Mitochondria are major sites of energy metabolism that influence numerous cellular events, including immunity and cancer development. Previously, we reported that the mitochondrion-specific antioxidant enzyme, manganese-containing superoxide dismutase (MnSOD), has dual roles in early- and late-carcinogenesis stages. However, how defective MnSOD impacts the chain of events that lead to cell transformation in pathologically normal epidermal cells that have been exposed to carcinogens is unknown. Here, we show that UVB radiation causes nitration and inactivation of MnSOD leading to mitochondrial injury and mitophagy. In keratinocytes, exposure to UVB radiation decreased mitochondrial oxidative phosphorylation, increased glycolysis and the expression of autophagy-related genes, and enhanced AKT Ser/Thr kinase (AKT) phosphorylation and cell growth. Interestingly, UVB initiated a prosurvival mitophagy response by mitochondria-mediated reactive oxygen species (ROS) signaling via the mammalian target of the mTOR complex 2 (mTORC2) pathway. Knockdown of rictor but not raptor abrogated UVB-induced mitophagy responses. Furthermore, fractionation and proximity-ligation assays reveal that ROS-mediated mTOC2 activation in mitochondria is necessary for UVB-induced mitophagy. Importantly, pretreatment with the MnSOD mimic MnTnBuOE-2-PyP5+ (MnP) attenuates mTORC2 activation and suppresses UVB-induced mitophagy. UVB radiation exposure also increased cell growth as assessed by soft-agar colony survival and cell growth assays, and pretreatment with MnP or the known autophagy inhibitor 3-methyladenine abrogated UVB-induced cell growth. These results indicate that MnSOD is a major redox regulator that maintains mitochondrial health and show that UVB-mediated MnSOD inactivation promotes mitophagy and thereby prevents accumulation of damaged mitochondria.

Triggering Mitophagy with Photosensitizers.

One can utilize light illumination to stimulate mitochondrial reactive oxygen species production through the use of mitochondria-specific photosensitizers. By proper tuning of the light dosage, the methodology permits probing of a multitude of mitochondrial damage responses, including mitophagy. This light-controllable trick offers unique opportunities for the investigation of mitophagy-one can spatiotemporally define mitochondrial damage, alter the number of impaired mitochondria, as well as modulate the severity of the mitochondrial injury. This light-activated mitophagy can be adapted not only to single-cell imaging techniques but also to cell population-based biochemical assays.

Temozolomide-Perillyl alcohol conjugate impairs Mitophagy flux by inducing lysosomal dysfunction in non-small cell lung Cancer cells and sensitizes them to irradiation.

BACKGROUND: Temozolomide-perillyl alcohol conjugate (TMZ-POH), a novel Temozolomide (TMZ) analog developed based on the conjugation of TMZ and perillyl alcohol (POH), displayed strong anticancer potency in multiple cancer types. In this study, we aimed to clarify the relationship between TMZ-POH and autophagy, and explore the underlying mechanisms involved in. METHODS: The proteins involved in autophagy, mitochondrial fission, lysosomal function and membrane traffic were detected by western blots; Autophagosome, mitochondria and lysosome were visualized by transmission electron microscope (TEM) and immunostaining; Apoptosis analysis and fluorescence probe detection were applied by flow cytometry. RESULTS: TMZ-POH blocked mitophagy flux although the number of autophagosomes which colocalized with mitochondria in the cells was increased via inducing lysosomal dysfunction as evidence from impaired lysosomal acidification, maturation and hampered autophagosome- lysosome fusion, which largely depended on its downregulation on the small GTPase RAB7A via mevalonate pathway. More importantly, our data demonstrated TMZ-POH sensitized cancer cell to irradiation induced apoptosis. CONCLUSIONS: Temozolomide-perillyl alcohol conjugate impairs mitophagy flux by inducing lysosomal dysfunction in Non-Small Cell Lung Cancer (NSCLC) cells and sensitizes them to irradiation, thereby proposing TMZ-POH as a potential radiosensitizer.

Regulation of Chlorophagy during Photoinhibition and Senescence: Lessons from Mitophagy.

Light energy is essential for photosynthetic energy production and plant growth. Chloroplasts in green tissues convert energy from sunlight into chemical energy via the electron transport chain. When the level of light energy exceeds the capacity of the photosynthetic apparatus, chloroplasts undergo a process known as photoinhibition. Since photoinhibition leads to the overaccumulation of reactive oxygen species (ROS) and the spreading of cell death, plants have developed multiple systems to protect chloroplasts from strong light. Recent studies have shown that autophagy, a system that functions in eukaryotes for the intracellular degradation of cytoplasmic components, participates in the removal of damaged chloroplasts. Previous findings also demonstrated an important role for autophagy in chloroplast turnover during leaf senescence. In this review, we describe the turnover of whole chloroplasts, which occurs via a type of autophagy termed chlorophagy. We discuss a possible regulatory mechanism for the induction of chlorophagy based on current knowledge of photoinhibition, leaf senescence and mitophagy-the autophagic turnover of mitochondria in yeast and mammals.

Fragmentation level determines mitochondrial damage response and subsequently the fate of cancer cells exposed to carbon ions.

OBJECTIVES: Although mitochondria are known to play an important role in radiation-induced cellular damage response, the mechanisms of how radiation elicits mitochondrial responses are largely unknown. MATERIALS AND METHODS: Human cervical cancer cell line HeLa and human breast cancer cell lines MCF-7 and MDA-MB-231 were irradiated with high LET carbon ions at low (0.5 Gy) and high (3 Gy) doses. Mitochondrial functions, dynamics, mitophagy, intrinsic apoptosis and total apoptosis, and survival fraction were investigated after irradiation. RESULTS: We found that carbon ions irradiation induced two different mitochondrial morphological changes and corresponding responses in cancer cells. Cells exposed to carbon ions of 0.5 Gy exhibited only modestly truncated mitochondria, and subsequently damaged mitochondria could be eliminated through mitophagy. In contrast, mitochondria within cells insulted by 3 Gy radiation split into punctate and clustered ones, which were associated with apoptotic cell death afterward. Inhibition of mitochondrial fission by Drp1 or FIS1 knockdown or with the Drp1 inhibitor mdivi-1 suppressed mitophagy and potentiated apoptosis after irradiation at 0.5 Gy. However, inhibiting fission led to mitophagy and increased cell survival when cells were irradiated with carbon ions at 3 Gy. CONCLUSION: We proposed a stress response model to provide a mechanistic explanation for the mitochondrial damage response to high-LET carbon ions.

Mitochondrial reactive oxygen species-mediated genomic instability in low-dose irradiated human cells through nuclear retention of cyclin D1.

Mitochondria are associated with various radiation responses, including adaptive responses, mitophagy, the bystander effect, genomic instability, and apoptosis. We recently identified a unique radiation response in the mitochondria of human cells exposed to low-dose long-term fractionated radiation (FR). Such repeated radiation exposure inflicts chronic oxidative stresses on irradiated cells via the continuous release of mitochondrial reactive oxygen species (ROS) and decrease in cellular levels of the antioxidant glutathione. ROS-induced oxidative mitochondrial DNA (mtDNA) damage generates mutations upon DNA replication. Therefore, mtDNA mutation and dysfunction can be used as markers to assess the effects of low-dose radiation. In this study, we present an overview of the link between mitochondrial ROS and cell cycle perturbation associated with the genomic instability of low-dose irradiated cells. Excess mitochondrial ROS perturb AKT/cyclin D1 cell cycle signaling via oxidative inactivation of protein phosphatase 2A after low-dose long-term FR. The resulting abnormal nuclear accumulation of cyclin D1 induces genomic instability in low-dose irradiated cells.

Severe mitochondrial damage associated with low-dose radiation sensitivity in ATM- and NBS1-deficient cells.

Low-dose radiation risks remain unclear owing to a lack of sufficient studies. We previously reported that low-dose, long-term fractionated radiation (FR) with 0.01 or 0.05 Gy/fraction for 31 d inflicts oxidative stress in human fibroblasts due to excess levels of mitochondrial reactive oxygen species (ROS). To identify the small effects of low-dose radiation, we investigated how mitochondria respond to low-dose radiation in radiosensitive human ataxia telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)1-deficient cell lines compared with corresponding cell lines expressing ATM and NBS1. Consistent with previous results in normal fibroblasts, low-dose, long-term FR increased mitochondrial mass and caused accumulation of mitochondrial ROS in ATM- and NBS1-complemented cell lines. Excess mitochondrial ROS resulted in mitochondrial damage that was in turn recognized by Parkin, leading to mitochondrial autophagy (mitophagy). In contrast, ATM- and NBS1-deficient cells showed defective induction of mitophagy after low-dose, long-term FR, leading to accumulation of abnormal mitochondria; this was determined by mitochondrial fragmentation and decreased mitochondrial membrane potential. Consequently, apoptosis was induced in ATM- and NBS1-deficient cells after low-dose, long-term FR. Antioxidant N-acetyl-L-cysteine was effective as a radioprotective agent against mitochondrial damage induced by low-dose, long-term FR among all cell lines, including radiosensitive cell lines. In conclusion, we demonstrated that mitochondria are target organelles of low-dose radiation. Mitochondrial response influences radiation sensitivity in human cells. Our findings provide new insights into cancer risk estimation associated with low-dose radiation exposure.

The Protective Roles of ROS-Mediated Mitophagy on 125I Seeds Radiation Induced Cell Death in HCT116 Cells.

For many unresectable carcinomas and locally recurrent cancers (LRC), 125I seeds brachytherapy is a feasible, effective, and safe treatment. Several studies have shown that 125I seeds radiation exerts anticancer activity by triggering DNA damage. However, recent evidence shows mitochondrial quality to be another crucial determinant of cell fate, with mitophagy playing a central role in this control mechanism. Herein, we found that 125I seeds irradiation injured mitochondria, leading to significantly elevated mitochondrial and intracellular ROS (reactive oxygen species) levels in HCT116 cells. The accumulation of mitochondrial ROS increased the expression of HIF-1α and its target genes BINP3 and NIX (BINP3L), which subsequently triggered mitophagy. Importantly, 125I seeds radiation induced mitophagy promoted cells survival and protected HCT116 cells from apoptosis. These results collectively indicated that 125I seeds radiation triggered mitophagy by upregulating the level of ROS to promote cellular homeostasis and survival. The present study uncovered the critical role of mitophagy in modulating the sensitivity of tumor cells to radiation therapy and suggested that chemotherapy targeting on mitophagy might improve the efficiency of 125I seeds radiation treatment, which might be of clinical significance in tumor therapy.

[Degradation of Mitochondria to Lipofuscin upon Heating and Illumination].

In the present work, it has been shown that the isolated mitochondria can undergo transformation to lipofuscin granules without any additional factors (oxygen saturation, prooxidants). The process occurs spontaneously and slowly at low temperature, and rapidly--by heating (thermo-lipofuscin) or under UV irradiation (photo-lipofuscin). The main contribution to the formation of mitochondrial lipofuscin comes from denatured proteins. Thermo-formation of lipofuscin depends on lipid peroxidation, while the presence of lipids is not required for photo-lipofuscin formation. It is shown that the use of detergent able to degrade mitochondria is necessary to measure lipofuscin content properly.