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Mitophagy Selectively Degrades Individual Damaged Mitochondria After Photoirradiation
Abstract
Damaged and dysfunctional mitochondria are proposed to be removed by autophagy. However, selective degradation of damaged mitochondria by autophagy (mitophagy) has yet to be experimentally verified. In this study, we investigated the cellular fate of individual mitochondria damaged by photoirradiation in hepatocytes isolated from transgenic mice expressing green fluorescent protein fused to microtubule-associated protein 1 light chain 3, a marker of forming and newly formed autophagosomes. Photoirradiation with 488-nm light induced mitochondrial depolarization (release of tetramethylrhodamine methylester [TMRM]) in a dose-dependent fashion. At lower doses of light, mitochondria depolarized transiently with re-polarization within 3 min. With greater light, mitochondrial depolarization became irreversible. Irreversible, but not reversible, photodamage induced autophagosome formation after 32±5 min. Photodamage-induced mitophagy was independent of TMRM, as photodamage also induced mitophagy in the absence of TMRM. Photoirradiation with 543-nm light did not induce mitophagy. As revealed by uptake of LysoTracker Red, mitochondria weakly acidified after photodamage before a much stronger acidification after autophagosome formation. Photodamage-induced mitophagy was not blocked by phosphatidylinositol 3-kinase inhibition with 3-methyladenine (10 mM) or wortmannin (100 nM). In conclusion, individual damaged mitochondria become selectively degraded by mitophagy, but photodamage-induced mitophagic sequestration occurs independently of the phosphatidylinositol 3-kinase signaling pathway, the classical upstream signaling pathway of nutrient deprivation-induced autophagy.
... Mitophagy is a selective autophagy in mitochondrial damages [8]. The common feature of mitophagy is the form of an autophagic vacuole enclosing damaged mitochondria, which is also well-defined as a mitophagosome [9]. ...
... Therefore, the regulation of autophagy is crucial for the safeguarding of heart homeostasis. Mitophagy is a selective autophagy in mitochondrial damages [8]. The common feature of mitophagy is the form of an autophagic vacuole enclosing damaged mitochondria, which is also well-defined as a mitophagosome [9]. ...
It has been proposed that procedures which upregulate mitochondrial biogenesis and autophagy by replacing damaged mitochondria with healthy ones may prevent the development of several heart diseases. A member of serine and threonine kinases, adenosine monophosphate-activated protein kinase (AMPK), could play essential roles in the autophagy and/or mitophagy. AMPK is widely distributed in various cells, which might play diverse regulatory roles in different tissues and/or organs. In fact, changes in the kinase function of AMPK due to alteration of activity have been linked with diverse pathologies including cardiac disorders. AMPK can regulate mitochondrial biogenesis via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) signaling and also improve oxidative mitochondrial metabolism through inhibition of mechanistic/mammalian target of rapamycin (mTOR) pathway, which may also modulate the autophagy/mitophagy through autophagy activating kinase 1 (ULK1) and/or transforming growth factor beta (TGF-β) signaling. Therefore, the modulation of AMPK in autophagy/mitophagy pathway might probably be thought as a therapeutic tactic for several cardiac disorders. As kinases are amongst the most controllable proteins, in general, the design of small molecules targeting kinases might be an eye-catching avenue to modulate cardiac function. Some analyses of the molecular biology underlying mitophagy suggest that nutraceuticals and/or drugs including specific AMPK modulator as well as physical exercise and/or dietary restriction that could modulate AMPK may be useful against several heart diseases. These observations may virtually be limited to preclinical studies. Come to think of these, however, it is speculated that some nutraceutical regimens might have positive potential for managing some of cardiac disorders.
... While the paradigm of oxidationinduced CaMKII activation is well established, [13][14][15] the current study examines CaMKII activation via autophosphorylation in relation to intracellular calcium modulation during sublethal Dox administration. 10,16,17 Mitophagy is regulated by calcium modulation 18 and is essential for maintaining mitochondrial function, playing both adaptive 19,20 and potentially maladaptive 6 roles under stress conditions. An increase in intramitochondrial calcium ([Ca 2+ ] mito) can destabilize the mitochondrial membrane potential (MMP) and induce cell death. ...
... 7,19 This response to stress could be interpreted as an adaptive mechanism to counterbalance Dox-induced alterations in mitochondrial dynamics. 20 Our findings indicate that the inhibition of pCAMKII by KN-93 significantly reduced Dox-induced (E) Ru360 pre-treatment failed to prevent the Dox-induced MMP decline, with MMP data measured via a microplate reader and presented as the R/G ratio (n = 7). Data are expressed as mean ± SD; scale bar = 100 µm. ...
Background Doxorubicin (Dox) is effective against different types of cancers, but it poses cardiotoxic side effects, frequently resulting in irreversible heart failure. However, the complexities surrounding this cardiotoxicity, especially at sublethal dosages, remain to be fully elucidated. We investigated early cellular disruptions in response to sublethal Dox, with a specific emphasis on the role of phosphorylated calcium/calmodulin-dependent protein kinase II (CaMKII) in initiating mitochondrial dysfunction.
Methods This study utilized the H9c2 cardiomyocyte model to identify a sublethal concentration of Dox and investigate its impact on mitochondrial health using markers such as mitochondrial membrane potential (MMP), mitophagy initiation, and mitochondrial calcium dynamics. We examined the roles of and interactions between CaMKII, dynamin-related protein 1 (Drp1), and the mitochondrial calcium uniporter (MCU) in Dox-induced mitochondrial disruption using specific inhibitors, such as KN-93, Mdivi-1, and Ru360, respectively.
Results Exposure to a sublethal dose of Dox reduced the MMP red-to-green fluorescence ratio in H9c2 cells by 40.6% compared with vehicle, and increased the proportion of cells undergoing mitophagy from negligible levels compared with vehicle to 62.2%. Mitochondrial calcium levels also increased by 8.7-fold compared with the vehicle group. Notably, the activation of CaMKII, particularly its phosphorylated form, was pivotal in driving these mitochondrial changes, as inhibition using KN-93 restored MMP and decreased mitophagy. However, inhibition of Drp1 and MCU functions had a limited impact on the observed mitochondrial disruptions.
Conclusion Sublethal administration of Dox is closely linked to CaMKII activation through phosphorylation, emphasizing its pivotal role in early mitochondrial disruption. These findings present a promising direction for developing therapeutic strategies that may alleviate the cardiotoxic effects of Dox, potentially increasing its clinical efficacy.
... Fluorescent-based strategies can provide a more robust assessment. These approaches often label mitochondria by autophagy marker microtubuleassociated protein light chain 3 (LC3), along with fluorescent proteins such as GFP-LC3 (Kim and Lemasters, 2011) or other mitochondrial fluorescent markers, such as TMRM and MFFR (Kim and Lemasters, 2011), mitochondrialtargeted RFP (Rambold et al., 2011), MitoTracker dyes (Dagda et al., 2009), and flag epitope-tagged Par (Kawajiri et al., 2010) and visualize colocalization of mitochondria enclosed in GFP-LC3-labeled autophagosomes. However, this approach has a relatively high false positive rate because LC3 proteins may aggregate in an autophagy-independent manner and easily incorporate into intracellular protein aggregates (Kuma et al., 2007). ...
... Fluorescent-based strategies can provide a more robust assessment. These approaches often label mitochondria by autophagy marker microtubuleassociated protein light chain 3 (LC3), along with fluorescent proteins such as GFP-LC3 (Kim and Lemasters, 2011) or other mitochondrial fluorescent markers, such as TMRM and MFFR (Kim and Lemasters, 2011), mitochondrialtargeted RFP (Rambold et al., 2011), MitoTracker dyes (Dagda et al., 2009), and flag epitope-tagged Par (Kawajiri et al., 2010) and visualize colocalization of mitochondria enclosed in GFP-LC3-labeled autophagosomes. However, this approach has a relatively high false positive rate because LC3 proteins may aggregate in an autophagy-independent manner and easily incorporate into intracellular protein aggregates (Kuma et al., 2007). ...
Mitochondria play an essential role in neural function, such as supporting normal energy metabolism, regulating reactive oxygen species, buffering physiological calcium loads, and maintaining the balance of morphology, subcellular distribution, and overall health through mitochondrial dynamics. Given the recent technological advances in the assessment of mitochondrial structure and functions, mitochondrial dysfunction has been regarded as the early and key pathophysiological mechanism of cognitive disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, and postoperative cognitive dysfunction. This review will focus on the recent advances in mitochondrial medicine and research methodology in the field of cognitive sciences, from the perspectives of energy metabolism, oxidative stress, calcium homeostasis, and mitochondrial dynamics (including fission-fusion, transport, and mitophagy).
... This loop can ultimately result in severe nuclear DNA damage and cell death [153]. Mitophagy degrades mitochondria with damaged DNA [154] and enhances longevity in rodent models [155]. However, the autophagy rate declines with age and chronic alcohol intake [53], promoting the accumulation of mtDNA mutations and a decline in mitochondrial function [156]. ...
... In type 2 mitophagy, cup-shaped phagophores are not formed; rather, LC3 aggregates sequester individual mitochondria into mitophagosomes. In both types of mitophagy, mitophagosomes form, acidify, fuse with lysosomes, and degrade their contents [154,156]. While type 1 is primarily related to physiological mechanisms, such as nutrient deprivation, type 2 is related to (and activated by) sensors of mitochondrial damage, such as those caused by oxidative stress [203]. ...
Ethanol consumption triggers oxidative stress by generating reactive oxygen species (ROS) through its metabolites. This process leads to steatosis and liver inflammation, which are critical for the development of alcoholic liver disease (ALD). Autophagy is a regulated dynamic process that sequesters damaged and excess cytoplasmic organelles for lysosomal degradation and may counteract the harmful effects of ROS-induced oxidative stress. These effects include hepatotoxicity, mitochondrial damage, steatosis, endoplasmic reticulum stress, inflammation, and iron overload. In liver diseases, particularly ALD, macroautophagy has been implicated as a protective mechanism in hepatocytes, although it does not appear to play the same role in stellate cells. Beyond the liver, autophagy may also mitigate the harmful effects of alcohol on other organs, thereby providing an additional layer of protection against ALD. This protective potential is further supported by studies showing that drugs that interact with autophagy, such as rapamycin, can prevent ALD development in animal models. This systematic review presents a comprehensive analysis of the literature, focusing on the role of autophagy in oxidative stress regulation, its involvement in organ–organ crosstalk relevant to ALD, and the potential of autophagy-targeting therapeutic strategies.
... Our work establishes that mitochondrial deletions can expand in muscle fibres without a replicative advantage and, surprisingly, even if mutants are preferentially eliminated from the system. Despite being subject to higher rates of mitophagy -something that finds experimental support [36,37,38,39,40,41] -mutants can come to dominate zones of the fibres. We also predict that the expansion of deletions takes place through a travelling wave of mutants propagating in the muscle fibres. ...
... In this study, we have modelled the accumulation of mutation during ageing through a bottomup, physically interpretable stochastic model based on Lotka-Volterra dynamics, one of the simplest classic models of population genetics. Our aim has been to explain why mitochondrial deletions clonally expand despite possibly being preferentially eliminated through quality control mechanisms [36,37,38,39,40]. Ours is the first spatial model to account for the spread of mutant mtDNA, schematising skeletal muscle fibres as a chain of nuclei each surrounded by a population of mitochondria that can move diffusively along the fibre. ...
The expansion of deleted mitochondrial DNA (mtDNA) molecules has been linked to ageing, particularly in skeletal muscle fibres; its mechanism has remained unclear for three decades. Previous accounts assigned a replicative advantage to the deletions, but there is evidence that cells can, instead, selectively remove defective mtDNA. We present a spatial model that, without a replicative advantage, but instead through a combination of enhanced density for mutants and noise, produces a wave of expanding mutations with wave speed consistent with experimental data, unlike a standard model based on replicative advantage. We provide a formula that predicts that the wave speed drops with copy number, in agreement with experimental data. Crucially, our model yields travelling waves of mutants even if mutants are preferentially eliminated. Justified by this exemplar of how noise, density and spatial structure affect muscle ageing, we introduce the mechanism of stochastic survival of the densest, an alternative to replicative advantage, that may underpin other phenomena, like the evolution of altruism.
... Mitophagy is a selective autophagy in mitochondrial damages [8]. The common feature of mitophagy is the form of autophagic vacuole enclosing damaged mitochondria, which is also well-2 defined as a mitophagosome [9]. ...
It has been proposed that procedures which up-regulate mitochondrial biogenesis and autophagy by replacing damaged mitochondria with healthy ones, may prevent the development of several heart diseases. A member of serine and threonine kinases, AMPK, could play essential roles in the autophagy and/or mitophagy. AMPK is widely distributed in various cells, which might play diverse regulatory roles in different tissues and/or organs. In fact, changes in the kinase function of AMPK due to alteration of activity have been linked with diverse pathologies including cardiac disorders. AMPK can regulate mitochondrial biogenesis via PGC1α signaling and also improve oxidative mitochondrial metabolism through inhibition of mTOR pathway, which may also modulate the autophagy/mitophagy through ULK1 and/or TGF-β signaling. Therefore, the modulation of AMPK in autophagy/mitophagy pathway might be considered as a therapeutic tactic for several cardiac disorders. As kinases are amongst the most controllable proteins, in general, the design of small molecules targeting kinases might be an eye-catching avenue to modulate the cardiac function. Some analyses of the molecular biology underlying mitophagy suggests that nutraceuticals and/or drugs including specific AMPK modulator as well as physical exercise and/or dietary restriction that could modulate AMPK may be useful against several heart diseases. In particular, some nutraceutical regimens may have encouraging potential for controlling some of cardiac disorders.
... Similar studies have treated MSCs with paclitaxel (PTX), commonly used in breast cancer treatment, where it was suggested that PTX-containing secretome demonstrates a pronounced inhibitory effect on the survival, migration and tumorigenicity of the triple-negative breast cancer (TNBC) cells [40]. Furthermore, as PI3K/Akt pathway is the classical upstream signaling pathway of nutrient deprivationinduced autophagy [41], Wort was able to inhibit autophagy, where it reduced the level of the autophagy marker (LC3II), relative to its basal level in control cells. However, loading MSCs secretome with 100 or 250 nM Wort enhanced the autophagy. ...
The utilization of Mesenchymal stem cells (MSCs)-derived secretome was suggested as a promising alternative in cell-based regenerative therapy. Herein, the MSCs cells were impregnated with a pan-PI3K/Akt/mTOR inhibitor and their secretome was utilized to explore the anticancer and antimetastasis effects against breast cancer. To establish this aim Bone marrow-derived MSCs was treated with 50, 100, or 250 nM Wortmannin (Wort), where the cytotoxic, apoptotic, and autophagic potential of their secretome were investigated in luminal-A breast cancer cells (MCF-7). We found that exposure of MCF-7 to Wort-containing secretome induced both apoptosis and autophagy, whereas prolonged exposure led to massive cell death. Also, Wort-loaded secretome induced nuclear DNA fragmentation and reduced cell metastasis in vitro. These findings were associated with Wort-dependent decrease in the formation of the phosphorylated Akt and mTOR proteins, reduced the expression of their mRNAs, and downregulate of the expression of the catalytic subunit of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K-CA). Taken together, these findings suggested the promising antiproliferative and antimetastasis effects of combining pan-PI3K/Akt/mTOR inhibitors with MSCs-derived secretome in breast cancer.
... Cells under photosensitive oxidative attack face intense redox stress and rely on autophagy and mitophagy to maintain the survival or replenishment of cells within the skin [67,68]. Mitophagy removes oxidized and depolarized mitochondria and prevents the release of proapoptotic proteins, the production of ROS, and the futile hydrolysis of ATP [69,70]. The self-saving process of senescence and autophagy of skin-resident cells may be linked through the MAPK pathway [71]. ...
Photoaging, the primary cause of exogenous skin aging and predominantly caused by ultraviolet radiation, is an essential type of skin aging characterized by chronic skin inflammation. Recent studies have shown that oxidative stress, inflammation, skin barrier homeostasis, collagen denaturation and pigmentation are the main contributors to it. As a composite tissue rich in matrix and vascular components, adipose tissue derivatives have been recently gaining attention as potential therapeutic agents for various human diseases with fat-processing technology upgrades. This review analyzes both ‘minimally treated’ and ‘nonminimally treated’ fat derivatives to give an overview of the preclinical and clinical relevance of adipose tissue derivatives for antiphotoaging application, highlighting their good clinical prospects as well as discussing their safety and potential risks.
... Given the non-regenerative nature of neurons, aberrant mitochondrial dynamics and metabolism can cause severe damage to the brain and even the entire nervous system. Numerous studies have uncovered an essential role for mitochondria in the pathogenesis of neurological diseases via different pathways and mechanisms (Kim and Lemasters, 2011;Lou et al., 2020;Lin et al., 2021;Mito et al., 2022). Accordingly, eukaryotic cells, including neural cells, have developed complex mechanisms to eliminate and degrade damaged mitochondria, with the most efficient being mitophagy (Kanki et al., 2009;Harper et al., 2018), which is activated by either ubiquitination or receptor pathways (Palikaras et al., 2018;Pfanner et al., 2019;Zhao et al., 2021). ...
Proper mitochondrial performance is imperative for the maintenance of normal neuronal function to prevent the development of neurodegenerative diseases. Persistent accumulation of damaged mitochondria plays a role in prion disease pathogenesis, which involves a chain of events that culminate in the generation of reactive oxygen species and neuronal death. Our previous studies have demonstrated that PINK1/Parkin-mediated mitophagy induced by PrP ¹⁰⁶⁻¹²⁶ is defective and leads to an accumulation of damaged mitochondria after PrP ¹⁰⁶⁻¹²⁶ treatment. Externalized cardiolipin (CL), a mitochondria-specific phospholipid, has been reported to play a role in mitophagy by directly interacting with LC3II at the outer mitochondrial membrane. The involvement of CL externalization in PrP ¹⁰⁶⁻¹²⁶ -induced mitophagy and its significance in other physiological processes of N2a cells treated with PrP ¹⁰⁶⁻¹²⁶ remain unknown. We demonstrate that the PrP ¹⁰⁶⁻¹²⁶ peptide caused a temporal course of mitophagy in N2a cells, which gradually increased and subsequently decreased. A similar trend in CL externalization to the mitochondrial surface was seen, resulting in a gradual decrease in CL content at the cellular level. Inhibition of CL externalization by knockdown of CL synthase, responsible for de novo synthesis of CL, or phospholipid scramblase-3 and NDPK-D, responsible for CL translocation to the mitochondrial surface, significantly decreased PrP ¹⁰⁶⁻¹²⁶ -induced mitophagy in N2a cells. Meanwhile, the inhibition of CL redistribution significantly decreased PINK1 and DRP1 recruitment in PrP ¹⁰⁶⁻¹²⁶ treatment but had no significant decrease in Parkin recruitment. Furthermore, the inhibition of CL externalization resulted in impaired oxidative phosphorylation and severe oxidative stress, which led to mitochondrial dysfunction. Our results indicate that CL externalization induced by PrP ¹⁰⁶⁻¹²⁶ on N2a cells plays a positive role in the initiation of mitophagy, leading to the stabilization of mitochondrial function.
... 24 Autophagy limits endogenous apoptosis caused by mitochondrial outer membrane permeabilization (MOMP) by removing damaged mitochondria. 45 Autophagy also selectively removes caspase-8, a key factor involved in exogenous apoptosis, to delay the occurrence of exogenous apoptosis. 46 In addition, autophagy selectively removes SRC tyrosine kinases to delay anoikis (anchorage-dependent cell death) induced by detachment from the extracellular matrix (ECM). ...
Jin-yan Dong,1 Hong-Lin Yin,2 Hao Hao,3 Yang Liu3 1First Clinical Medical College, Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 2Faculty of Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China; 3Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of ChinaCorrespondence: Hao Hao; Yang Liu, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan, People’s Republic of China, Tel +86-13583119291 ; +86-13864018185, Email haohao0826@163.com; 13864018185@163.comAbstract: Autophagy is a highly conserved process that maintains cell stability in eukaryotes, participates in the turnover of intracellular substances to maintain cell function, helps to resist pathogen invasion, and improves cell tolerance to environmental changes. Autophagy has been observed in many diseases, and the symptoms of these diseases are significantly improved by regulating autophagy. Autophagy is also involved in the development of lung diseases. Studies have shown that autophagy may play a beneficial or harmful role in acute lung injury (ALI), and ALI has been treated with traditional Chinese medicine designed to promote or inhibit autophagy. In this paper, the molecular mechanism and common pathways regulating autophagy and the relationship between autophagy and ALI are introduced, and the active ingredients of traditional Chinese medicine that improve ALI symptoms by regulating autophagy are summarized.Keywords: autophagy, inflammation, oxidative stress, apoptosis
... PINK1 is a serine/ threonine kinase with an N-terminal mitochondrial targeting sequence. The PINK1-dependent pathway is one of the most-studied mitophagy mechanisms [63][64][65][66]. Accumulation of PINK1 in dysfunctional mitochondria can recruit Parkin, OPTN, NDP52, and other binding partners that, in turn, induce the degradation of the damaged mitochondria through mitophagy. ...
Sterile α and toll/interleukin 1 receptor motif-containing protein 1 (SARM1) is the defining molecule and central executioner of programmed axon death, also known as Wallerian degeneration. SARM1 has a mitochondrial targeting sequence, and it can bind to and stabilize PTEN-induced putative kinase 1 (PINK1) for mitophagy induction, but the deletion of the mitochondrial localization sequence is found to disrupt the mitochondrial localization of SARM1 in neurons without altering its ability to promote axon degeneration after axotomy. The biological significance of SARM1 mitochondrial localization remains elusive. In this study, we observed that the pro-degeneration factor, SARM1, was upregulated in acrylamide (ACR) neuropathy, a slow, Wallerian-like, programmed axonal death process. The upregulated SARM1 accumulated on mitochondria, interfered with mitochondrial dynamics, and activated PINK1-mediated mitophagy. Importantly, rapamycin (RAPA) intervention eliminated mitochondrial accumulation of SARM1 and partly attenuated ACR neuropathy. Thus, mitochondrial localization of SARM1 may contribute to its clearance through the SARM1-PINK1 mitophagy pathway, which inhibits axonal degeneration through a negative feedback loop. The mitochondrial localization of SARM1 complements the coordinated activity of the pro-survival factor, nicotinamide mononucleotide adenyltransferase 2 (NMNAT2), and SARM1 and is part of the self-limiting molecular mechanisms underpinning programmed axon death in ACR neuropathy.
Graphical abstract
Mitophagy clearance of SARM1 is complementary to the coordinated activity of NMNAT2 and SARM1 in ACR neuropathy.
... Within the fusion-fission cycle, selection can occur during fusion through factors that increase or decrease the likelihood that mutant mitochondria are included. There is also good evidence that selection occurs after fission through different likelihoods of wild-type and mutant mitochondria being degraded via mitophagy (23)(24)(25)(26)(27). ...
Mitochondria are cellular organelles of crucial relevance for the survival of metazoan organisms. Damage to the mitochondrial DNA can give rise to a variety of mitochondrial diseases and is thought also to be involved in the aging process. The fate of mtDNA mutants is controlled by their synthesis as well as degradation and mathematical models can help to better understand this complex interplay. We present here a model that combines a replicative advantage for mtDNA mutants with selective degradation enabled by mitochondrial fission and fusion processes. The model shows not only that the cell has efficient means to deal with (many) types of mutants, but surprisingly it also predicts that under certain conditions a stable co-existence of mutant and wild type mtDNAs is possible. We discuss how this new finding might explain how mitochondria can be at the heart of processes with such different phenotypes as mitochondrial diseases and aging.
... Phagosomes swallow malfunctioning and old mitochondria during mitophagy and eliminate them from biological systems (Lemasters, 2014). Depending on the type of trigger, mitophagy is divided into three categories: type 1 mitophagy is triggered by a lack of nutrients, type 2 is triggered by damage signals, and type 3 is triggered by tiny mitochondrial-derived vesicles (Kim and Lemasters, 2011). Although mitophagy is well known for removing damaged mitochondria, the method through which mitochondria are recruited for mitophagy remains unknown. ...
Neurons depend on mitochondrial functions for membrane excitability, neurotransmission, and plasticity. Mitochondrial dynamics are important for neural cell maintenance. To maintain mitochondrial homeostasis, lysosomes remove dysfunctional mitochondria through mitophagy. Mitophagy promotes mitochondrial turnover and prevents the accumulation of dysfunctional mitochondria. In many neurodegenerative diseases (NDDs), including Alzheimer's disease (AD), mitophagy is disrupted in neurons. Mitophagy is regulated by several proteins; recently, Rho-associated coiled-coil containing protein kinase 2 (ROCK2) has been suggested to negatively regulate the Parkin-dependent mitophagy pathway. Thus, ROCK2 inhibition may be a promising therapy for NDDs. This review summarizes the mitophagy pathway, the role of ROCK2 in Parkin-dependent mitophagy regulation, and mitophagy impairment in the pathology of AD. We further discuss different ROCK inhibitors (synthetic drugs, natural compounds, and gene therapy-based approaches) and examine their effects on triggering neuronal growth and neuroprotection in AD and other NDDs. This comprehensive overview of the role of ROCK in mitophagy inhibition provides a possible explanation for the significance of ROCK inhibitors in the therapeutic management of AD and other NDDs.
... Mitochondria are highly dynamic, energy generating organelles that are known to concentrate in regions of high energy demand [11,12] and are densely packed in sensory nerve terminal boutons [13,14]. Mitochondrial membrane potential (MMP) is a marker of optimal mitochondrial function where mitochondrial depolarization can indicate mitochondrial dysfunction [15][16][17]. Depolarization and the loss of MMP impacts respiratory chain complexes, which interrupts cellular electron flow and results in ATP depletion [18][19][20][21]. Maintenance of MMP is fundamental for the normal performance and survival of cells that have a high-energy requirement [22], such as sensory neurons [23][24][25]. ...
Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M 1 R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M 1 R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M 1 R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M 1 R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M 1 R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain, and enhance nerve repair in peripheral neuropathy.
... In addition, mitochondrial DNA mutations are also associated with aging (Cree et al. 2008). As the number of mutations in mitochondria exceeds the critical minimum, they are eliminated, decreasing their per-cell copy numbers (Kim and Lemasters 2011;Gaziev et al. 2014). The main energy source of ATP for M2 macrophages is mitochondrial oxidative phosphorylation. ...
Atherosclerosis is a systemic autoimmune disease of the arterial wall characterized by chronic inflammation, high blood pressure, oxidative stress, and progressive loss of cell and organ function with aging. An imbalance of macrophage polarization is associated with many aging diseases, including atherosclerosis. The polarization toward the pro-inflammatory M1 macrophage is a major promoter of the atheroma formation. It is known that efferocytosis, or ingestion of apoptotic cells, is stimulated by M2 macrophage polarization. A failure of efferocytosis leads to the prolongation of chronic pathology in tissue. In addition, fat-laden macrophages contribute to the plague progression by transforming into foam cells in response to excess lipid deposition in arteries. In spite of the generally accepted theory that macrophages capture oxidized low-density lipoprotein by phagocytosis and become foam cells, we postulate that the main source of lipid accumulation in foam cells are senescent erythrocytes. Senescent erythrocytes lose their plasticity, which affects the rheological blood properties. It is known that their membrane contains high levels of cholesterol. There is evidence that senescent erythrocytes play a pathogenic role in the atheroma formation after breaking down during flowing through an artery bifurcation. Here we review the current knowledge on the impact of age-associated immune cells and red blood cells modifications on atherogenesis.
Graphical abstract :
... Mitophagy can be prevented by a dominant-negative mutant of Drp1, suggesting that fission is required for mobilizing such cellular degeneration (32). Damaged mitochondria undergo selective mitophagy (33), which is also consistent with the fission event as it elicits quality control by segregating the damaged mitochondria and eliminating them through autophagy. Mitophagy is usually regulated by one of the following three pathways: FUNDC1, BNIP3/NIP3-Nix pathway (hypoxiainduced), or PINK1/Parkin mediated pathway (non-hypoxia induced) (34). ...
Significance
Inositol pyrophosphates are versatile messenger molecules containing the energetic pyrophosphate bond. One of the principal enzymes generating the inositol pyrophosphate IP 7 (5-diphosphoinositolpentakisphosphate) is inositol hexakisphosphate kinase 2 (IP6K2). Previous work has shown that IP6K2 is neuroprotective and maintains mitochondrial respiration. We now report that loss of IP6K2 leads to increased mitochondrial fission and mitophagy. Regulation of mitochondrial dynamics by IP6K2 depends on the protein PINK1 and, interestingly, is independent of IP6K2 enzymatic activity. These findings provide mechanistic insight into the regulation of mitochondrial function by IP6K2, which has implications for neuroprotection and mitochondrial physiology more generally.
... In contrast, mitochondrial depolarization/damage precedes and initiates autophagic sequestration in Type 2 mitophagy [25,26,53]. Ordinarily, functional mitochondria import fulllength PINK1 using the mitochondrial protein import machinery and driven by membrane potential (ΔΨ). ...
Ethanol increases hepatic mitophagy driven by unknown mechanisms. Type 1 mitophagy sequesters polarized mitochondria for nutrient recovery and cytoplasmic remodeling. In Type 2, mitochondrial depolarization (mtDepo) initiates mitophagy to remove the damaged organelles. Previously, we showed that acute ethanol administration produces reversible hepatic mtDepo. Here, we tested the hypothesis that ethanol-induced mtDepo initiates Type 2 mitophagy. GFP-LC3 transgenic mice were gavaged with ethanol (2-6 g/kg) with and without pre-treatment with agents that decrease or increase mtDepo-Alda-1, tacrolimus, or disulfiram. Without ethanol, virtually all hepatocytes contained polarized mitochondria with infrequent autophagic GFP-LC3 puncta visualized by intravital microscopy. At ~4 h after ethanol treatment, mtDepo occurred in an all-or-none fashion within individual hepatocytes, which increased dose dependently. GFP-LC3 puncta increased in parallel, predominantly in hepatocytes with mtDepo. Mitochondrial PINK1 and PRKN/parkin also increased. After covalent labeling of mitochondria with MitoTracker Red (MTR), GFP-LC3 puncta encircled MTR-labeled mitochondria after ethanol treatment, directly demonstrating mitophagy. GFP-LC3 puncta did not associate with fat droplets visualized with BODIPY558/568, indicating that increased autophagy was not due to lipophagy. Before ethanol administration, rhodamine-dextran (RhDex)-labeled lysosomes showed little association with GFP-LC3. After ethanol treatment, TFEB (transcription factor EB) translocated to nuclei, and lysosomal mass increased. Many GFP-LC3 puncta merged with RhDex-labeled lysosomes, showing autophagosomal processing into lysosomes. After ethanol treatment, disulfiram increased, whereas Alda-1 and tacrolimus decreased mtDepo, and mitophagy changed proportionately. In conclusion, mtDepo after acute ethanol treatment induces mitophagic sequestration and subsequent lysosomal processing.Abbreviations : AcAld, acetaldehyde; ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ALD, alcoholic liver disease; Alda-1, N-(1,3-benzodioxol-5-ylmethyl)-2,6-dichlorobenzamide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; LAMP1, lysosomal-associated membrane protein 1; LMNB1, lamin B1; MAA, malondialdehyde-acetaldehyde adducts; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; MPT, mitochondrial permeability transition; mtDAMPS, mitochondrial damage-associated molecular patterns; mtDepo, mitochondrial depolarization; mtDNA, mitochondrial DNA; MTR, MitoTracker Red; PI, propidium iodide; PINK1, PTEN induced putative kinase 1; PRKN, parkin; RhDex, rhodamine dextran; TFEB, transcription factor EB; Tg, transgenic; TMRM, tetramethylrhodamine methylester; TOMM20, translocase of outer mitochondrial membrane 20; VDAC, voltage-dependent anion channel.
... And mitophagy, a speci c type of autophagy that selectively degrades defective mitochondria, has received extra attention in energy maintenance for damaged axons. PINK1 dependent pathway is one of the beststudied mitophagy mechanisms [52][53][54][55]. PINK1 is a serine/threonine kinase with an N-terminal mitochondrial targeting sequence. ...
Sterile-α and toll/interleukin 1 receptor motif containing protein 1 (SARM1) is the central executioner of programmed axon death (Wallerian degeneration). Although it has been confirmed to have a mitochondrial targeting sequence and can bind to and stabilize PINK1 on mitochondria, the biological significance for mitochondrial localization of SARM1 is still unclear. The relationship between mitochondrial quality control mechanisms and programmed axon death also needs to be clarified. Chronic acrylamide (ACR) intoxication cause typical pathology of axon degeneration involving early axon loss. Here, we demonstrated that the SARM1 dependent Wallerian axon self-destruction pathway was activated following ACR intoxication. Moreover, increased SARM1 was observed on the mitochondria, which interfered with the mitochondrial quality control mechanisms. As a protective response to stress, mitochondrial components enriched in SARM1 were isolated from the mitochondrial network through an increased fission process and were degraded in an autophagy-dependent manner. Importantly, rapamycin (RAPA) administration eliminated mitochondrial accumulated SARM1 and inhibited axon loss. Thus, mitochondrial localization of SARM1 may be complement to the coordinated activity of NMNAT2 and SARM1, and may be part of the self-limiting molecular mechanisms of programmed axon death. In the early latent period, the mitochondrial localization of SARM1 will help it to be isolated by the mitochondrial network and to be degraded through mitophagy to maintain local axon homeostasis. When the mitochondrial quality control mechanisms are broken down, SARM1 will cause irreversible damage for axon death.
... Radiation-induced mitochondrial dysfunction and biogenesis are known to be related to mitochondrial autophagy [201]. Under conditions of extensive mitochondrial damage, the cell undergoes mitophagy so as to eliminate the damaged and dysfunctional mitochondria. ...
Population aging is occurring rapidly worldwide, challenging the global economy and healthcare services. Brain aging is a significant contributor to various age-related neurological and neuropsychological disorders, including Alzheimer's disease and Parkinson's disease. Several ex-trinsic factors, such as exposure to ionizing radiation, can accelerate senescence. Multiple human and animal studies have reported that exposure to ionizing radiation can have varied effects on organ aging and lead to the prolongation or shortening of life span depending on the radiation dose or dose rate. This paper reviews the effects of radiation on the aging of different types of brain cells, including neurons, microglia, astrocytes, and cerebral endothelial cells. Further, the relevant molecular mechanisms are discussed. Overall, this review highlights how radiation-induced senescence in different cell types may lead to brain aging, which could result in the development of various neurological and neuropsychological disorders. Therefore, treatment targeting radiation-induced oxidative stress and neuroinflammation may prevent radiation-induced brain aging and the neuro-logical and neuropsychological disorders it may cause.
... Mitophagy is another mechanism that tumor cells usually use as a rescue cellular homeostasis to compensate for mitochondrial photodamage of PDT. Mitophagy is a selective type of autophagy that functions as a negative regulatory feedback mechanism that reduces the mitochondrial-derived ROS production and prevents the release of proapoptotic proteins to abort cell death [34][35][36]. Nonetheless, excessive ROS generation of PDT causes the recruitment of ubiquitin ligase PRKN/parkin to initiate the degradation of the mitochondria through a process called mitophagy [34,37]. ...
Photodynamic therapy (PDT) is currently enjoying considerable attention as the subject of experimental research to treat resistant cancers. The preferential accumulation of a non-toxic photosensitizer (PS) in different cellular organelles that causes oxidative damage by combining light and molecular oxygen leads to selective cell killing. However, one major setback, common among other treatment approaches, is tumor relapse and the development of resistance causing treatment failure. PDT-mediated resistance could result from increased drug efflux and decreased localization of PS, reduced light exposure, increased DNA damage repair, and altered expression of survival genes. This review highlights the essential insights of PDT reports in which PDT resistance was observed and which identified some of the molecular effectors that facilitate the development of PDT resistance. We also discuss different perceptions of PDT and how its current limitations can be overturned to design improved cancer resistant treatments.
... Mitophagy is a specific form of autophagy that selectively clears damaged mitochondria. 69,70 Autophagy induction (e.g., by nutrient deprivation, sulforaphane treatment, etc.) can result in mitochondria elongation, inhibiting the release of cytochrome c 71 , which prevents apoptosis and sustains cell viability. 72 Apoptosis induction following cytochrome c release activates 76 Alternatively, in mitochondrial apoptosis deficient cells, autophagy induction can eventually result in stress-induced cell death through caspase-8 activation. ...
... Mitophagy, as a selective autophagy in mitochondrial damage, is first identified in yeast [22]. The common feature Figure 1: Mitochondrial dysfunctions involved in cardiac disease. ...
The normal function of the mitochondria is crucial for most tissues especially for those that demand a high energy supply. Emerging evidence has pointed out that healthy mitochondrial function is closely associated with normal heart function. When these processes fail to repair the damaged mitochondria, cells initiate a removal process referred to as mitophagy to clear away defective mitochondria. In cardiomyocytes, mitophagy is closely associated with metabolic activity, cell differentiation, apoptosis, and other physiological processes involved in major phenotypic alterations. Mitophagy alterations may contribute to detrimental or beneficial effects in a multitude of cardiac diseases, indicating potential clinical insights after a close understanding of the mechanisms. Here, we discuss the current opinions of mitophagy in the progression of cardiac diseases, such as ischemic heart disease, diabetic cardiomyopathy, cardiac hypertrophy, heart failure, and arrhythmia, and focus on the key molecules and related pathways involved in the regulation of mitophagy. We also discuss recently reported approaches targeting mitophagy in the therapy of cardiac diseases.
... On the other side, however, numerous studies indicate that pharmacological or genetic inhibition of autophagy greatly enhances cell death [21][22][23]. It is generally agreed that autophagy is a pro-survival mechanism by not only providing nutrients for cell survival during starvation but also selectively removing damaged organelles, including damaged mitochondria [70]. Liver-specific knockout of Atg7 leads to hepatomegaly and severe liver injury [23] and suppression of autophagy exacerbates alcohol-induced liver injury by increasing alcohol induced apoptosis [24]. ...
Background
Alcoholic liver disease (ALD) is the most common liver disease worldwide and its underlying molecular mechanisms are still poorly understood. Moreover, conflicting data have been reported on potentially protective autophagy, the exact role of ethanol-metabolizing enzymes and ROS.
Methods
Expression of LC3B, CYP2E1, and NOX4 was studied in a mouse model of acute ethanol exposure by immunoblotting and immunohistochemistry. Autophagy was further studied in primary mouse hepatocytes and huh7 cells in response to ethanol and its major intermediator acetaldehyde. Experiments were carried out in cells overexpressing CYP2E1 and knock down of NOX4 using siRNA. The response to external H2O2 was studied by using the GOX/CAT system. Autophagic flux was monitored using the mRFP-GFP-LC3 plasmid, while rapamycin and chloroquine served as positive and negative controls.
Results
Acute ethanol exposure of mice over 24 hours significantly induced autophagy as measured by LC3B expression but also induced the ROS-generating CYP2E1 and NOX4 enzymes. Notably, ethanol but not its downstream metabolite acetaldehyde induced autophagy in primary mouse hepatocytes. In contrast, autophagy could only be induced in huh7 cells in the presence of overexpressed CYP2E1. In addition, overexpression of NOX4 also significantly increased autophagy, which could be blocked by siRNA mediated knock down. The antioxidant N-acetylcysteine (NAC) also efficiently blocked CYP2E1-and NOX4-mediated induction of autophagy. Finally, specific and non-toxic production of H2O2 by the GOX/CAT system as evidenced by elevated peroxiredoxin (Prx-2) also induced LC3B which was efficiently blocked by NAC. H2O2 strongly increased the autophagic flux as measured by mRFP-GFP-LC3 plasmid.
Conclusion
We here provide evidence that short-term ethanol exposure induces autophagy in hepatocytes both in vivo and in vitro through the generation of ROS. These data suggest that suppression of autophagy by ethanol is most likely due to longer alcohol exposure during chronic alcohol consumption with the accumulation of e.g. misfolded proteins.
... The mice modle showed disruption of autophagy caused loss of mitochondrial activity and generation of ROS [50]. Additionally inflammasomes are also detected in the damaged mitochondria due to autophagy blockage [51,52]. Elmore and coworkers reported that NPs induced mitochondrial damage induces autophagy blockage and inactivation persists until the removal of cellular debris from cell [53]. ...
Autophagy is defined as a process in which cell undergo sequential sequestration of degraded protein and damaged organelles for maintenance of cellular hemostasis. Autophagy is regulated by complex mammalian target of rapamycin complex 1 (m-TORC) pathway, and its types depends on nature phagocytized material. Interaction of autophagic signaling pathway with nanocarriers causes autophagy inhibition and cell death. The role different classes of nanomaterials in autophagy perturbation, both regulation and inhibition in in-vitro and animal models were studied. These nanocarriers have application in anticancer delivery agents to cancer cells. Due to retention of NPs in mitochondria and endoplasmic reticulum induces stress and up-regulates autophagy sequences. This co-localization of NPs in organelles lead to cancer cell death due to autophagy inhibition. The review covers autophagy regulation pathways and NPs mediated autophagy modulation in combination with chemotherapeutics has been explored for cancer therapy with their toxicity concerns.
... As above-discussed, the UV-radiation induced mtDNA damages cannot be repaired by mtDNA repair mechanisms. However, the mitochondria with photo-damaged mtDNA undergoes mitophagy (Kim and Lemasters, 2011). It was confirmed that mitophagy can be induced by different types of mtDNA damage stimuli, and it at least partially depends on DNA damage response pathways (Furda et al., 2012). ...
Mitochondria are double membrane organelles in eukaryotic cells that provide energy by generating adenosine triphosphate (ATP) through oxidative phosphorylation. They are crucial to many aspects of cellular metabolism. Mitochondria contain their own DNA that encodes for essential proteins involved in the execution of normal mitochondrial functions. Compared with nuclear DNA, the mitochondrial DNA (mtDNA) is more prone to be affected by DNA damaging agents, and accumulated DNA damages may cause mitochondrial dysfunction and drive the pathogenesis of a variety of human diseases, including neurodegenerative disorders and cancer. Therefore, understanding better how mtDNA damages are repaired will facilitate developing therapeutic strategies. In this review, we focus on our current understanding of the mtDNA repair system. We also discuss other mitochondrial events promoted by excessive DNA damages and inefficient DNA repair, such as mitochondrial fusion, fission, and mitophagy, which serve as quality control events for clearing damaged mtDNA.
... Therefore, in healthy mitochondria, the levels of PINK1 are low in cells. Upon stresses such as mitochondrial depolarization [143], impairment of protein import [144], and others [145][146][147], PINK1 import into mitochondria is blocked and as a result, it accumulates on the OMM [148]. Once there, it binds the mitochondrial TOM complex [149,150] and it is activated by auto-phosphorylation. Its activation leads to a signaling cascade by recruiting and activating other proteins like Parkin [143,151] and Ubiquitin (Ub) [152]. ...
Mitochondria are organelles central to myriad cellular processes. To maintain mitochondrial health, various processes co-operate at both the molecular and organelle level. At the molecular level, mitochondria can sense imbalances in their homeostasis and adapt to these by signaling to the nucleus. This mito-nuclear communication leads to the expression of nuclear stress response genes. Upon external stimuli, mitochondria can also alter their morphology accordingly, by inducing fission or fusion. In an extreme situation, mitochondria are degraded by mitophagy. Adequate function and regulation of these mitochondrial quality control pathways are crucial for cellular homeostasis. As we discuss, alterations in these processes have been linked to several pathologies including neurodegenerative diseases and cancer.
... Mitophagy is a systemic culling of mitochondria by autophagy (137), and increases when cells are exposed to acute cytotoxic stressors (67,(138)(139)(140)(141). A seminal finding was that mitochondrial fission events separated metabolically healthy from unhealthy daughter organelles with subsequent removal of the damaged daughter via mitophagy (35). ...
Mitochondrial fission protein 1 (Fis1) was identified in yeast as being essential for mitochondrial division or fission and subsequently determined to mediate human mitochondrial and peroxisomal fission. Yet, its exact functions in humans, especially in regard to mitochondrial fission, remains an enigma as genetic deletion of Fis1 elongates mitochondria in some cell types, but not others. Fis1 has also been identified as an important component of apoptotic and mitophagic pathways suggesting the protein may have multiple, essential roles. This review presents current perspectives on the emerging functions of Fis1 and their implications in human health and diseases, with an emphasis on Fis1’s role in both endocrine and neurological disorders.
... The first mechanism is by upregulating mitophagy. Mitophagy is induced by the loss of mitochondrial membrane potential (∆Ψm), leading to increased inner membrane permeability and the ubiquitylation of multiple outer membrane proteins such as voltage-dependent anion-selective channel 1 (VDAC1), mitofusin 1 (MFN1) and MFN2 promoting mitophagy [87][88][89]. Mitophagy induction has been observed by an increasing accumulation of P62 in the pre-ischemic cortex of a mouse model of transient middle cerebral artery occlusion (MCAO) treated with rapamycin [90]. Thus, rapamycin could attenuate ischemic brain injury via the induction of mitophagy. ...
Simple Summary
Autophagy is a self-eating mechanism that is involved in the degradation of organelles and cellular materials. It is initiated by intracellular and extracellular stress stimuli. In the context of tumor development, microenvironmental hypoxic stress regulates autophagy that, in turn, promotes cancer-cell death or cancer-cell survival. Autophagy functions and shares molecular players with other cell-death promoting pathways such as apoptosis. Here, we discuss the spatial and temporal control of autophagy that could result in opposing cellular outcomes. We also address the role of immune cells polarization in this context. This knowledge is essential for efficiently targeting autophagy in conjunction with immunotherapy for improved cancer treatment.
Abstract
Programmed cell death or type I apoptosis has been extensively studied and its contribution to the pathogenesis of disease is well established. However, autophagy functions together with apoptosis to determine the overall fate of the cell. The cross talk between this active self-destruction process and apoptosis is quite complex and contradictory as well, but it is unquestionably decisive for cell survival or cell death. Autophagy can promote tumor suppression but also tumor growth by inducing cancer-cell development and proliferation. In this review, we will discuss how autophagy reprograms tumor cells in the context of tumor hypoxic stress. We will illustrate how autophagy acts as both a suppressor and a driver of tumorigenesis through tuning survival in a context dependent manner. We also shed light on the relationship between autophagy and immune response in this complex regulation. A better understanding of the autophagy mechanisms and pathways will undoubtedly ameliorate the design of therapeutics aimed at targeting autophagy for future cancer immunotherapies.
... Depending on the extent of mitochondrial photodamage, tumor cells elicit mitophagy to rescue cellular homeostasis through clearance of oxidized or depolarized mitochondria. Mitophagy has several distinct variants (i.e., type 1, 2, and 3) and prevents the release of proapoptotic proteins, generation of toxic mitochondrial-derived ROS, and futile ATP hydrolysis (137)(138)(139)(140). A primary cellular response following the mitochondrial photodamage is the recruitment of the E3 ubiquitin ligase PRKN/parkin to the mitochondrial outer membrane, which depends on PINK1 (59,141). ...
Cancer is considered an age-related disease that, over the next 10 years, will become the most prevalent health problem worldwide. Although cancer therapy has remarkably improved in the last few decades, novel treatment concepts are needed to defeat this disease. Photodynamic Therapy (PDT) signalize a pathway to treat and manage several types of cancer. Over the past three decades, new light sources and photosensitizers (PS) have been developed to be applied in PDT. Nevertheless, there is a lack of knowledge to explain the main biochemical routes needed to trigger regulated cell death mechanisms, affecting, considerably, the scope of the PDT. Although autophagy modulation is being raised as an interesting strategy to be used in cancer therapy, the main aspects referring to the autophagy role over cell succumbing PDT-photoinduced damage remain elusive. Several reports emphasize cytoprotective autophagy, as an ultimate attempt of cells to cope with the photo-induced stress and to survive. Moreover, other underlying molecular mechanisms that evoke PDT-resistance of tumor cells were considered. We reviewed the paradigm about the PDT-regulated cell death mechanisms that involve autophagic impairment or boosted activation. To comprise the autophagy-targeted PDT-protocols to treat cancer, it was underlined those that alleviate or intensify PDT-resistance of tumor cells. Thereby, this review provides insights into the mechanisms by which PDT can be used to modulate autophagy and emphasizes how this field represents a promising therapeutic strategy for cancer treatment.
... It is proposed that autophagosome membranes may derive from endoplasmic reticulum (ER) [11][12][13], mitochondria [14,15], the Golgi apparatus [16,17], the recycling endosomes [18][19][20], and plasma membrane [21]. In addition to non-selective sequestration of cellular components usually induced by nutritional deprivation, increasing evidence has suggested that autophagy can also be responsible for the removal of specific cellular materials, such as protein aggregates (aggrephagy), aberrant mitochondria (mitophagy), pathogens (xenophagy) and other superfluous organelles [22][23][24][25][26]. The mechanisms of selective autophagy are intricate and not yet fully understood. ...
Cancer cell expression of PD-L1 leads to T cells exhaustion by transducing co-inhibitory signal, and further understanding the regulation of PD-L1 in cancer cells may provide additional therapeutic strategies. Here by drug repurposing screen, we identified amlodipine as a potent inhibitor of PD-L1 expression in cancer cells. Further survey of calcium-associated pathways revealed calpain-dependent stabilization of the PD-L1 protein. Intracellular calcium delivered an operational signal to calpain-dependent Beclin-1 cleavage, blocking autophagic degradation of PD-L1 accumulated on recycling endosome (RE). Blocking calcium flux by amlodipine depleted PD-L1 expression and increased CD8+ T-cell infiltration in tumor tissues but not in myocardium, causing dose-dependent tumor suppression in vivo. Rescuing PD-L1 expression eliminated the effects of amlodipine, suggesting the PD-L1-dependent effect of amlodipine. These results reveal a calcium-dependent mechanism controlling PD-L1 degradation, and highlight calcium flux blockade as a potential strategy for combinatorial immunotherapy.
... i. L'autophagie protège de la mort cellulaire L'autophagie inhibe l'apoptose en particulier grâce à la dégradation sélective de composants essentiels à l'induction de l'apoptose. En effet, les mitochondries qui ont subi une perte de leur potentiel transmembranaire et une fragmentation mitochondriale subissent la mitophagie (autophagie sélective des mitochondries) (Kim and Lemasters, 2011;Youle and Narendra, 2011). De plus, il a été décrit que la caspase 8 active peut également être la cible de l'autophagie sélective, et être envoyée vers la dégradation lysosomale (Hou et al., 2010). ...
Les G-quadruplexes (G4) sont des structures non canoniques des acides nucléiques qui peuvent être formés dans des régions d’ADN ou d’ARN riches en guanines. Les ligands G4 (LG4), sont des molécules capables d’interagir et de stabiliser les structures G4, qui présentent de nombreuses propriétés anti-cancéreuses. Nous avons travaillé avec le LG4 20A, appartenant à la famille des triarylpyridines, qui stabilise efficacement les structures G4 in vitro. Les objectifs de ce travail ont été de déterminer les mécanismes moléculaires et cellulaires responsables des effets anti-prolifératifs du 20A dans des cellules cancéreuses. Dans cette étude, nous avons montré que le 20A induit un arrêt de la croissance cellulaire de cellules en culture et dans un modèle de xénogreffe tumorale, grâce à l’induction de la sénescence et de la mort cellulaire par apoptose. Ces réponses sont associées à l’activation de la voie des réponses aux dommages à l’ADN (DDR) via la kinase ATM, qui favorise l’autophagie (un processus catabolique) et la sénescence, tout en protégeant les cellules de l’apoptose. De plus, nous avons observé que le 20A induit un échec de la cytokinèse, conduisant à l’accumulation de cellules binucléées qui présentent une résistance à la mort cellulaire. De façon inattendue, nous avons trouvé que le 20A s’accumule dans les lysosomes, induisant une augmentation de la taille de ces derniers. La combinaison du 20A et de l’agent lysomotropique chloroquine, potentialise de façon importante la perméabilisation de la membrane lysosomale (LMP) et la mort cellulaire. En particulier, cette combinaison sensibilise de façon notable ces cellules binucléées à la mort cellulaire. L’ensemble de ces résultats révèle une relation entre les processus de mort cellulaire et de sénescence induits par le LG4 20A, et les voies de DDR et lysosomales. Ces régulations devraient être prises en considération lors de l’utilisation d’agents antiprolifératifs susceptibles d’interférer avec les fonctions lysosomales.
... As damaged mitochondria promote the production of harmful reactive oxygen species (ROS) and stimulate inflammation, cells have developed specialized quality control mechanisms to eliminate damaged/depolarized mitochondria. These function by either selectively removing damaged mitochondrial proteins using mitochondria derived vesicles or completely eliminating depolarized mitochondria by a selective autophagy mechanism, termed mitophagy, that requires mitochondrial fission to selectively deliver damaged mitochondria to lysosomes and prevent damaging effects on cells (Twig et al., 2008;Kim and Lemasters, 2011;Soubannier et al., 2012;Shirihai et al., 2015;Fritsch et al., 2020). ...
SARS-CoV-2 is a positive sense RNA coronavirus that constitutes a new threat for the global community and economy. While vaccines against SARS-CoV-2 are being developed, the mechanisms through which this virus takes control of an infected cell to replicate remains poorly understood. Upon infection, viruses completely rely on host cell molecular machinery to survive and replicate. To escape from the immune response and proliferate, viruses strategically modulate cellular metabolism and alter subcellular organelle architecture and functions. One way they do this is by modulating the structure and function of mitochondria, a critical cellular metabolic hub but also a key platform for the regulation of cellular immunity. This versatile nature of mitochondria defends host cells from viruses through several mechanisms including cellular apoptosis, ROS signaling, MAVS activation and mitochondrial DNA-dependent immune activation. These events are regulated by mitochondrial dynamics, a process by which mitochondria alter their structure (including their length and connectivity) in response to stress or other cues. It is therefore not surprising that viruses, including coronaviruses hijack these processes for their survival. In this review, we highlight how positive sense RNA viruses modulate mitochondrial dynamics and metabolism to evade mitochondrial mediated immune response in order to proliferate.
... Fluorescent LAMP1 can then be used in combination with mitochondrial probes to follow the later events of mitophagy [227]. Alternatively, LysoTracker dyes can also be used to follow the colocalization of mitochondria with lysosomes [224,228,229]. These dyes have acidotropic properties, and therefore, they are recruited to the acidic compartments in the cell [230]. ...
Mitochondria are multifunctional organelles that are crucial to cell homeostasis. They constitute the major site of energy production for the cell, they are key players in signalling pathways using secondary messengers such as calcium, and they are involved in cell death and redox balance paradigms. Mitochondria quickly adapt their dynamics and biogenesis rates to meet the varying energy demands of the cells, both in normal and in pathological conditions. Therefore, understanding simultaneous changes in mitochondrial functions is crucial in developing mitochondria-based therapy options for complex pathological conditions such as cancer, neurological disorders, and metabolic syndromes. To this end, fluorescence microscopy coupled to live imaging represents a promising strategy to track these changes in real time. In this review, we will first describe the commonly available tools to follow three key mitochondrial functions using fluorescence microscopy: Calcium signalling, mitochondrial dynamics, and mitophagy. Then, we will focus on how the development of genetically-encoded fluorescent sensors became a milestone for the understanding of these mitochondrial functions. In particular, we will show how these tools allowed researchers to address several biochemical activities in living cells, and with high spatiotemporal resolution. With the ultimate goal of tracking multiple mitochondrial functions simultaneously, we will conclude by presenting future perspectives for the development of novel genetically-encoded fluorescent biosensors.
... As damaged mitochondria promote the production of harmful reactive oxygen species (ROS) and stimulate inflammation, cells have developed specialized quality control mechanisms to eliminate damaged/depolarized mitochondria. These function by either selectively removing damaged mitochondrial proteins using mitochondria derived vesicles or completely eliminating depolarized mitochondria by a selective autophagy mechanism, termed mitophagy, that requires mitochondrial fission to selectively deliver damaged mitochondria to lysosomes and prevent damaging effects on cells (Twig et al., 2008;Kim and Lemasters, 2011;Soubannier et al., 2012;Shirihai et al., 2015;Fritsch et al., 2020). ...
SARS-CoV-2 is a positive sense RNA coronavirus that constitutes a new threat for the global community and economy. While vaccines against SARS-CoV-2 are being developed, the mechanisms through which this virus takes control of an infected cell to replicate remains poorly understood. Upon infection, viruses completely rely on host cell molecular machinery to survive and replicate. To escape from the immune response and proliferate, viruses strategically modulate cellular metabolism and alter subcellular organelle architecture and functions. One way they do this is by modulating the structure and function of mitochondria, a critical cellular metabolic hub but also a key platform for the regulation of cellular immunity. This versatile nature of mitochondria defends host cells from viruses through several mechanisms including cellular apoptosis, ROS signaling, MAVS activation and mitochondrial DNA-dependent immune activation. These events are regulated by mitochondrial dynamics, a process by which mitochondria alter their structure (including their length and connectivity) in response to stress or other cues. It is therefore not surprising that viruses, including coronaviruses hijack these processes for their survival. In this review, we highlight how positive sense RNA viruses modulate mitochondrial dynamics and metabolism to evade mitochondrial mediated immune response in order to proliferate.
Keratinocytes, located in the outermost layer of human skin, are pivotal cells to resist environmental damage. Cellular autophagy plays a critical role in eliminating damaged organelles and maintaining skin cell homeostasis. Low‐dose 5‐Aminolevulinic acid photodynamic therapy (ALA‐PDT) has been demonstrated to enhance skin's antistress ability; however, the regulatory mechanisms of autophagy in keratinocytes remain unclear. In this study, we treated immortalized human keratinocytes (HaCaT cells) with low‐dose ALA‐PDT (0.5 mmol/L, 3 J/cm ² ). Through RNA‐sequencing analysis, we identified that low‐dose ALA‐PDT modulated autophagy‐related pathways in keratinocytes and pinpointed Unc‐51‐like kinase 1 (ULK1) as a key gene involved. Western blot results revealed that low‐dose ALA‐PDT treatment upregulated the expression of autophagy‐related proteins Beclin‐1 and LC3‐II/LC3‐I ratio. Notably, low‐dose ALA‐PDT regulated autophagy by inducing an appropriate level of reactive oxygen species (ROS), transiently reducing mitochondrial membrane potential, and decreasing adenosine triphosphate production; all these processes functioned on the AMP‐activated protein kinase (AMPK)/ULK1 pathway to activate autophagy. Finally, we simulated external environmental damage using ultraviolet B (UVB) at a dose of 60 mJ/cm ² and observed that low‐dose ALA‐PDT mitigated UVB‐induced cell apoptosis; however, this protective effect was reversed when using the autophagy inhibitor 3‐methyladenine. Overall, these findings highlight how low‐dose ALA‐PDT enhances antistress ability in HaCaT cells through controlling ROS generation and activating the AMPK/ULK1 pathway to arouse cellular autophagy.
Cold atmospheric plasma (CAP) is a novel technology that generates a unique combination of reactive oxygen and nitrogen species (ROS/RNS), electric fields, and UV radiation. CAP has shown promise in regulating the immune system and has potential clinical applications in wound healing, cancer treatment, and infection control. This review provides an overview of the immunological regulation activity of CAP, highlighting its substantial impact on cytokines production, immune cell phagocytosis, and immune cell proliferation. CAP has also been demonstrated to have potent therapeutic effect in anti‐inflammation, wound repair, viral and bacterial infections. Furthermore, CAP has been investigated as an adjuvant therapy for tumor treatment, eliciting a robust antitumor immune response and remarkable synergistic effects in diverse combination therapies. Further research is needed to fully understand the mechanisms underlying the effects of CAP on the immune system and to optimize its clinical application.
The present review summarizes the beneficial and detrimental roles of reactive oxygen species in myocardial ischemia/reperfusion injury and cardioprotection. In the first part, the continued need for cardioprotection beyond that by rapid reperfusion of acute myocardial infarction is emphasized. Then, pathomechanisms of myocardial ischemia/reperfusion to the myocardium and the coronary circulation and the different modes of cell death in myocardial infarction are characterized. Different mechanical and pharmacological interventions to protect the ischemic/reperfused myocardium in elective percutaneous coronary interventions and coronary artery bypass grafting, in acute myocardial infarction and in cardiotoxicity from cancer therapy are detailed. The second part keeps the focus on ROS providing a comprehensive overview of molecular and cellular mechanisms involved in ischemia/reperfusion injury. Starting from mitochondria as the main sources and targets of ROS in ischemic/reperfused myocardium, a complex network of cellular and extracellular processes is discussed, including relationships with Ca²⁺ homeostasis, thiol group redox balance, hydrogen sulfide modulation, cross-talk with NAPDH oxidases, exosomes, cytokines and growth factors. While mechanistic insights are needed to improve our current therapeutic approaches, advancements in knowledge of ROS-mediated processes indicate that detrimental facets of oxidative stress are opposed by ROS requirement for physiological and protective reactions. This inevitable contrast is likely to underlie unsuccessful clinical trials and limits the development of novel cardioprotective interventions simply based upon ROS removal.
Mitochondria play a crucial role in cellular respiration, ATP production, and the regulation of various cellular processes. Mitochondrial dysfunctions have been directly linked to pathophysiological conditions, making them a significant target of interest in toxicological research. In recent years, there has been a growing need to understand the intricate effects of xenobiotics on human health, necessitating the use of effective scientific research tools. Caenorhabditis elegans (C. elegans), a nonpathogenic nematode, has emerged as a powerful tool for investigating toxic mechanisms and mitochondrial dysfunction. With remarkable genetic homology to mammals, C. elegans has been used in studies to elucidate the impact of contaminants and drugs on mitochondrial function. This review focuses on the effects of several toxic metals and metalloids, drugs of abuse and pesticides on mitochondria, highlighting the utility of C. elegans as a model organism to investigate mitochondrial dysfunction induced by xenobiotics. Mitochondrial structure, function, and dynamics are discussed, emphasizing their essential role in cellular viability and the regulation of processes such as autophagy, apoptosis, and calcium homeostasis. Additionally, specific toxins and toxicants, such as arsenic, cadmium, and manganese are examined in the context of their impact on mitochondrial function and the utility of C. elegans in elucidating the underlying mechanisms. Furthermore, we demonstrate the utilization of C. elegans as an experimental model providing a promising platform for investigating the intricate relationships between xenobiotics and mitochondrial dysfunction. This knowledge could contribute to the development of strategies to mitigate the adverse effects of contaminants and drugs of abuse, ultimately enhancing our understanding of these complex processes and promoting human health.
Purpose:
Fuchs endothelial corneal dystrophy (FECD) is characterized by an accelerated depletion of corneal endothelial cells. There is growing evidence that mitochondrial exhaustion is central in the pathology. Indeed, endothelial cells loss in FECD forces the remaining cells to increase their mitochondrial activity, leading to mitochondrial exhaustion. This generates oxidation, mitochondrial damage, and apoptosis, fueling a vicious cycle of cells' depletion. This depletion ultimately causes corneal edema and irreversible loss of transparency and vision. Concurrently to endothelial cells loss, the formation of extracellular mass called guttae on the Descemet's membrane, is a hallmark of FECD. The pathology origins at the center of the cornea and progress outward, like the appearance of guttae.
Methods:
Using corneal endothelial explants from patients with late-stage FECD at the time of their corneal transplantation, we correlated mitochondrial markers (mitochondrial mass, potential, and calcium) and the level of oxidative stress and apoptotic cells, with the area taken by guttae. The different markers have been analyzed using fluorescent-specific probes and microscopic analysis.
Results:
We observed a positive correlation between the presence of guttae and the level of mitochondrial calcium and apoptotic cells. We found a negative correlation between the presence of guttae and the level of mitochondrial mass, membrane potential, and oxidative stress.
Conclusions:
Taken together, these results show that the presence of guttae is correlated with negative outcome in the mitochondrial health, oxidative status, and survival of nearby endothelial cells. This study provides insight on FECD etiology that could lead to treatment targeting mitochondrial stress and guttae.
https://www.mdpi.com/books/book/6617-induced-impairment-of-neurogenesis-and-brain-diseases
Aim:
Liver transplantation (LT) is the only curative therapy for decompensated liver cirrhosis. For recipients of living donor LT (LDLT), restoration of liver function after transplantation is highly dependent on liver regenerative capacity, which requires large amounts of intracellular energy. Mitochondrial metabolism provides a stable supply of ATP for liver regeneration. Mitophagy is a selective process in which damaged, non-functional mitochondria are degraded and replaced with new functional mitochondria. We investigated the relationship between expression of Syntaxin17 (STX17), a key protein in mitophagy regulation, in donor livers and graft survival.
Methods:
We examined STX17 expression in grafts from 143 LDLT donors who underwent right lobe resection and investigated the relationship between STX17 expression and graft function. We investigated the correlations among STX17 expression, mitochondrial membrane potential and cell proliferation, using a STX17-knockdown hepatocyte cell line.
Results:
Recipients transplanted with low STX17-expression grafts had significantly lower graft survival rates than recipients transplanted with high STX17-expression grafts (88.9% vs. 100%, p<0.01). Multivariate analysis showed that low STX17 expression (HR: 10.7, CI: 1.29-88.0, p<0.05) and the absence of splenectomy (HR: 6.27, CI: 1.59-24.8, p<0.01) were independent predictive factors for small-for-size graft syndrome, which is the severe complication in LDLT. In the vitro experiments, the percentage of depolarized damaged mitochondria was increased in the STX17-knockdown hepatocyte cell line, suggesting decreased mitophagy and ATP synthesis. Cell proliferation was significantly decreased in the STX17-knockdown hepatocyte cell line.
Conclusion:
STX17 contributes to mitophagy and maintenance of mitochondrial function in hepatocytes and may be a predictor of graft dysfunction in LDLT patients. This article is protected by copyright. All rights reserved.
Impairments in mitochondrial physiology play a role in the progression of multiple neurodegenerative conditions, including peripheral neuropathy in diabetes. Blockade of muscarinic acetylcholine type 1 receptor (M 1 R) with specific/selective antagonists prevented mitochondrial dysfunction and reversed nerve degeneration in in vitro and in vivo models of peripheral neuropathy. Specifically, in type 1 and type 2 models of diabetes, inhibition of M 1 R using pirenzepine or muscarinic toxin 7 (MT7) induced AMP-activated protein kinase (AMPK) activity in dorsal root ganglia (DRG) and prevented sensory abnormalities and distal nerve fiber loss. The human neuroblastoma SH-SY5Y cell line has been extensively used as an in vitro model system to study mechanisms of neurodegeneration in DRG neurons and other neuronal sub-types. Here, we tested the hypothesis that pirenzepine or MT7 enhance AMPK activity and via this pathway augment mitochondrial function in SH-SY5Y cells. M 1 R expression was confirmed by utilizing a fluorescent dye, ATTO590-labeled MT7, that exhibits great specificity for this receptor. M 1 R antagonist treatment in SH-SY5Y culture increased AMPK phosphorylation and mitochondrial protein expression (OXPHOS). Mitochondrial membrane potential (MMP) was augmented in pirenzepine and MT7 treated cultured SH-SY5Y cells and DRG neurons. Compound C or AMPK-specific siRNA suppressed pirenzepine or MT7-induced elevation of OXPHOS expression and MMP. Moreover, muscarinic antagonists induced hyperpolarization by activating the M-current and, thus, suppressed neuronal excitability. These results reveal that negative regulation of this M 1 R-dependent pathway could represent a potential therapeutic target to elevate AMPK activity, enhance mitochondrial function, suppress neuropathic pain and enhance nerve repair in peripheral neuropathy.
As the fourth state of matter, plasma’s unique properties and interactions with other states of matter offer many promising opportunities for investigation and discovery. In particular, cold atmospheric plasma (CAP), operating at atmospheric pressure and room temperature, has remarkable potential for biomedical applications through various delivery methods. These biomedical applications include sterilization, wound healing, blood coagulation, oral/dental diseases treatment, cancer therapy, and immunotherapy. Effective delivery of plasma constituents is critical to its efficacy for these applications. Therefore, this review presents the key research activities related to CAP delivery (including direct CAP delivery, delivery of plasma-activated media, biomedical device-assisted plasma delivery, and CAP delivery with other therapeutics) and needs for future research. This review will be of great interest for understanding the current state-of-the-art of biomedical applications of plasma medicine while also giving researchers from a broad range of communities insight into research efforts that would benefit from their contributions. Such communities include biomedicine, physics, biochemistry, material science, nanotechnology, and medical device manufacturing.
Sterile-α and toll/interleukin 1 receptor motif containing protein 1 (SARM1) is the defining molecule and central executioner of programmed axon death (also known as Wallerian degeneration). Although it has been confirmed to have a mitochondrial targeting sequence and can bind to and stabilize PTEN-induced putative kinase 1 (PINK1) for mitophagy induction, deletion of the mitochondrial localization sequence disrupts SARM1 mitochondrial localization in neurons but does not alter its ability to promote axon degeneration after axotomy. The biological significance for mitochondrial localization of SARM1 remains elusive. Here, we demonstrated that the SARM1-dependent axonal destruction pathway was involved in acrylamide (ACR) neuropathy in vivo and in vitro , a moderate Wallerian-like programmed axonal death process. The up-regulated SARM1 accumulated on mitochondria, which interfered with mitochondrial dynamics and activated PINK1-mediated mitophagy. Importantly, rapamycin (RAPA) intervention eliminated mitochondrial accumulated SARM1 and partly attenuated ACR neuropathy. Thus, mitochondrial localization of SARM1 contributes to its clearance through the SARM1-PINK1 mitophagy pathway and mitophagy, in turn, negative feedback inhibits axonal degeneration. Mitochondrial localization of SARM1 is complementary to the coordinated activity of the pro-survival factor, nicotinamide mononucleotide adenyltransferase 2 (NMNAT2), and SARM1, and is part of the self-limiting molecular mechanisms of programmed axon death.
Background: Sterile-α and toll/interleukin 1 receptor motif containing protein 1 (SARM1) is the central executioner of axon degeneration. Although it has been confirmed to have a mitochondrial targeting sequence and can bind to and stabilize PINK1 on depolarized mitochondria, the biological significance for mitochondrial localization of SARM1 is still unclear. Chronic acrylamide (ACR) intoxication can cause typical pathology of axonal injury, owning the potential to explore the interaction between mitochondria and SARM1 during the latent period of axon destruction.
Methods: The expression and the mitochondria distribution of SARM1 were evaluated in in vivo and in vitro ACR neuropathy models. Transmission electron microscopy, immunoblotting, and immunofluorescence were performed to evaluate mitochondrial dynamics and PINK1-dependent mitophagy. LC3 turnover experiment and live cell imaging were conducted to further assess the state of mitophagy flux. In order to verify the effect of mitophagy in SARM1-mediated axon degeneration, low-dose and low-frequency rapamycin was administered in ACR-exposed rats to increase basal autophagy.
Results: In a time- and dose-dependent manner, ACR induced peripheral nerve injury in rats and truncated axons of differentiated N2a cell. Moreover, the severity of this axon damage was consistent with the up-regulation of SARM1. SARM1 prominently accumulated on mitochondria, and at the same time mitophagy was activated. Importantly, rapamycin (RAPA) administration eliminated mitochondrial accumulated SARM1 and alleviated SARM1 dependent axonal degeneration.
Conclusions: Complementing to the coordinated activity of NMNAT2 and SARM1, mitochondrial localization of SARM1 may be part of the self-limiting molecular mechanisms of Wallerian axon destruction. In the early latent period of axon damage, the mitochondrial localization of SARM1 will help it to be isolated by the mitochondrial network and to be degraded through PINK1-dependent mitophagy to maintain local axon homeostasis. When the mitochondrial quality control mechanisms are broken down, SARM1 will cause irreversible damage for axon degeneration. Moderate autophagy activation can be invoked as potential strategies to alleviate axon degeneration in ACR neuropathy and even other axon degeneration diseases.
Mitophagy is a selective form of macroautophagy in which dysfunctional and damaged mitochondria can be efficiently degraded, removed and recycled through autophagy. Selective removal of damaged or fragmented mitochondria is critical to the functional integrity of the entire mitochondrial network and cells. In past decades, numerous studies have shown that mitophagy is involved in various diseases; however, since the dual role of mitophagy in tumour development, mitophagy role in tumour is controversial, and further elucidation is needed. That is, although mitophagy has been demonstrated to contribute to carcinogenesis, cell migration, ferroptosis inhibition, cancer stemness maintenance, tumour immune escape, drug resistance, etc. during cancer progression, many research also shows that to promote cancer cell death, mitophagy can be induced physiologically or pharmacologically to maintain normal cellular metabolism and prevent cell stress responses and genome damage by diminishing mitochondrial damage, thus suppressing tumour development accompanying these changes. Signalling pathway-specific molecular mechanisms are currently of great biological significance in the identification of potential therapeutic targets. Here, we review recent progress of molecular pathways mediating mitophagy including both canonical pathways (Parkin/PINK1- and FUNDC1-mediated mitophagy) and noncanonical pathways (FKBP8-, Nrf2-, and DRP1-mediated mitophagy); and the regulation of these pathways, and abovementioned pro-cancer and pro-death roles of mitophagy. Finally, we summarise the role of mitophagy in cancer therapy. Mitophagy can potentially be acted as the target for cancer therapy by promotion or inhibition.
Cellular senescence, a stable cell division arrest caused by severe damage and stress, is a hallmark of aging in vertebrates including humans. With progressing age, senescent cells accumulate in a variety of mammalian tissues, where they contribute to tissue aging, identifying cellular senescence as a major target to delay or prevent aging. There is an increasing demand for the discovery of new classes of small molecules that would either avoid or postpone cellular senescence by selectively eliminating senescent cells from the body (i.e., ‘senolytics’) or inactivating/switching damage‐inducing properties of senescent cells (i.e., ‘senostatics/senomorphics’), such as the senescence‐associated secretory phenotype. Whereas compounds with senolytic or senostatic activity have already been described, their efficacy and specificity has not been fully established for clinical use yet. Here, we review mechanisms of senescence that are related to mitochondria and their interorganelle communication, and the involvement of proteostasis networks and metabolic control in the senescent phenotype. These cellular functions are associated with cellular senescence in in vitro and in vivo models but have not been fully exploited for the search of new compounds to counteract senescence yet. Therefore, we explore possibilities to target these mechanisms as new opportunities to selectively eliminate and/or disable senescent cells with the aim of tissue rejuvenation. We assume that this research will provide new compounds from the chemical space which act as mimetics of caloric restriction, modulators of calcium signaling and mitochondrial physiology, or as proteostasis optimizers, bearing the potential to counteract cellular senescence, thereby allowing healthy aging.
Cancer cell expression of PD-L1 leads to T cells exhaustion by transducing co-inhibitory signal, and further understanding the regulation of PD-L1 in cancer cells may provide additional therapeutic strategies. Here by drug repurposing screen we identified amlodipine as a potent inhibitor of PD-L1 expression in cancer cells. Further survey of calcium-associated pathways revealed calpains-dependent stabilization of the PD-L1 protein. Intracellular calcium delivered an operational signal to calpain-dependent Beclin-1 cleavage, blocking autophagic degradation of PD-L1 accumulated on recycling endosome (RE). Blocking calcium flux by amlodipine depleted PD-L1 expression and increased CD8+ T cell infiltration in tumor tissues but not in myocardium, causing dose-dependent tumor suppression in vivo. Rescuing PD-L1 expression eliminated the effects of amlodipine, suggesting the PD-L1 dependent effect of amlodipine. These results reveal a calcium-dependent mechanism controlling PD-L1 degradation, and highlight calcium flux blockade as a potential strategy for combinatorial immunotherapy.
Mitochondria play an essential role in oxidative phosphorylation, fatty acid oxidation, and the regulation of apoptosis. However, this organelle also produces reactive oxygen species (ROS) that continually inflict oxidative damage on mitochondrial DNA, proteins, and lipids, which causes further production of ROS. To oppose this oxidative stress, mitochondria possess quality control systems that include antioxidant enzymes and the repair or degradation of damaged mitochondrial DNA and proteins. If the oxidative stress exceeds the capacity of the mitochondrial quality control system, it seems that autophagy degrades the damaged mitochondria to maintain cellular homeostasis. Indeed, recent evidence from yeast to mammals indicates that the autophagy-dependent degradation of mitochondria (mitophagy) contributes to eliminate dysfunctional, aged, or excess mitochondria. In this paper, we describe the molecular processes and regulatory mechanisms of mitophagy in yeast and mammalian cells.
Inhibition of the TOR signalling pathway by genetic or pharmacological intervention extends lifespan in invertebrates, including yeast, nematodes and fruitflies; however, whether inhibition of mTOR signalling can extend lifespan in a mammalian species was unknown. Here we report that rapamycin, an inhibitor of the mTOR pathway, extends median and maximal lifespan of both male and female mice when fed beginning at 600 days of age. On the basis of age at 90% mortality, rapamycin led to an increase of 14% for females and 9% for males. The effect was seen at three independent test sites in genetically heterogeneous mice, chosen to avoid genotype-specific effects on disease susceptibility. Disease patterns of rapamycin-treated mice did not differ from those of control mice. In a separate study, rapamycin fed to mice beginning at 270 days of age also increased survival in both males and females, based on an interim analysis conducted near the median survival point. Rapamycin may extend lifespan by postponing death from cancer, by retarding mechanisms of ageing, or both. To our knowledge, these are the first results to demonstrate a role for mTOR signalling in the regulation of mammalian lifespan, as well as pharmacological extension of lifespan in both genders. These findings have implications for further development of interventions targeting mTOR for the treatment and prevention of age-related diseases.
Loss-of-function mutations in Park2, the gene coding for the ubiquitin ligase Parkin, are a significant cause of early onset Parkinson's disease. Although the role of Parkin in neuron maintenance is unknown, recent work has linked Parkin to the regulation of mitochondria. Its loss is associated with swollen mitochondria and muscle degeneration in Drosophila melanogaster, as well as mitochondrial dysfunction and increased susceptibility to mitochondrial toxins in other species. Here, we show that Parkin is selectively recruited to dysfunctional mitochondria with low membrane potential in mammalian cells. After recruitment, Parkin mediates the engulfment of mitochondria by autophagosomes and the selective elimination of impaired mitochondria. These results show that Parkin promotes autophagy of damaged mitochondria and implicate a failure to eliminate dysfunctional mitochondria in the pathogenesis of Parkinson's disease.
Electron microscopic morphometry has demonstrated a rapid decrease in the fractional volume of autophagic vacuoles (AV) in hepatocytes of adult male rats after the intraperitoneal administration of insulin (5 U/kg of body weight). Except for a significant decrease in glycogen to about one-half its initial value, no major changes in the composition of the remaining cytoplasm, or in the average volume of the single hepatocyte, were seen. The decrease found in the AVs is attributed to an inhibition of the formation of new AVs-probably the morphologic counterpart of the well-known anticatabolic effects of insulin. The decay of the fractional volume of the AVs appeared to follow first-order kinetics. Thus, the termination of the "life" of an AV by destruction of its contents may not depend directly on the "age" of the AV. The average half-life of the AVs amounted to approximately 9 min. Similar values were found for the different types of AVs, except for those containing glycogen. The half-life of these AVs was approximately 18 min. From the half-life values and from the "segregated fractions" at time zero, which were different for the different cytoplasmic components, rates of removal from the cytoplasm by autophagy were calculated. Expressed as "percent per day", the following rates were found: whole cytoplasm, 2.3; mitochondria, 3.9; microbodies, 8.9; and glycogen, 1.1. The results indicate that autophagy, to some extent, is selective and plays an important, but not an exclusive, role in intracellular turnover.
Seven cytosolic enzymes with varying half-lives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h; glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organelles (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeptin. Inhibitors of lysosomal fusion processes (vinblastine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolytic inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.
Among the most controversial hypotheses of aging are those which involve the progressive accumulation of error-bearing or altered macromolecules with advancing age. The effect of low levels of error or of alterations in only one or a small number of the many macromolecular mitochondrial components might be amplified by the highly integrated process of mitochondrial biosynthesis and observed as a change in turnover rates. The turnover rates of mitochondria from a variety of tissues of young adult (12-month-old) and aged (24-month-old) rats were measured by following the loss of radioactivity from proteins of purified mitochondrial preparations after initial labeling with ³H-leucine. Mitochondria from liver, brain, heart, and testes appeared to lose label as a homogeneous class with respect to rate. However, mitochondria from kidney, lung, and intestinal mucosa exhibited at least one additional exponential component. No significant differences were found for any tissue between the two age groups. Estimates for the half-lives in days obtained by combining both young and old sets of data are: liver, 9.3; testes, 12.6; heart, 17.5; brain 24.4; small intestine, 0.7 (first) and 17.6 (second); lung, 4 (first) and 16.6 (second); and kidney, 6 (first) and 10.9 (second). It is concluded that these data do not support the concept that errors in macromolecules are accumulated with age.
Tumor necrosis factor-alpha receptor 1 and Fas recruit overlapping signaling pathways. To clarify the differences between tumor necrosis factor alpha (TNFalpha) and Fas pathways in hepatocyte apoptosis, primary mouse hepatocytes were treated with TNFalpha or an agonist anti-Fas antibody after infection with an adenovirus expressing an IkappaB superrepressor (Ad5IkappaB). Treatment with TNFalpha induced apoptosis in Ad5IkappaB-infected mouse hepatocytes, as we previously reported for rat hepatocytes. Ad5IkappaB plus anti-Fas antibody or actinomycin D plus anti-Fas antibody rapidly induced apoptosis, whereas anti-Fas antibody alone produced little cytotoxicity. The proteasome inhibitor (MG-132) and a dominant-negative mutant of nuclear factor-kappaB-inducing kinase also promoted TNFalpha- and Fas-mediated apoptosis. Expression of either crmA or a dominant-negative mutant of the Fas-associated death domain protein prevented TNFalpha- and Fas-mediated apoptosis. In addition, the caspase inhibitors, DEVD-cho and IETD-fmk, inhibited TNFalpha- and Fas-mediated apoptosis. In Ad5IkappaB-infected hepatocytes, caspases-3 and -8 were activated within 2 h after treatment with anti-Fas antibody or within 6 h after TNFalpha treatment. Confocal microscopy demonstrated onset of the mitochondrial permeability transition (MPT) and mitochondrial depolarization by 2-3 h after anti-Fas antibody treatment and 8-10 h after TNFalpha treatment, followed by cytochrome c release. The combination of the MPT inhibitors, cyclosporin A, and trifluoperazine, protected Ad5IkappaB-infected hepatocytes from TNFalpha-mediated apoptosis. After anti-Fas antibody, cyclosporin A and trifluoperazine decreased cytochrome c release but did not prevent caspase-3 activation and cell-death. In conclusion, nuclear factor-kappaB activation protects mouse hepatocytes against both TNFalpha- and Fas-mediated apoptosis. TNFalpha and Fas recruit similar but nonidentical, pathways signaling apoptosis. The MPT is obligatory for TNFalpha-induced apoptosis. In Fas-mediated apoptosis, the MPT accelerates the apoptogenic events but is not obligatory for them.
Little is known about the protein constituents of autophagosome membranes in mammalian cells. Here we demonstrate that the rat microtubule-associated protein 1 light chain 3 (LC3), a homologue of Apg8p essential for autophagy in yeast, is associated to the autophagosome membranes after processing. Two forms of LC3, called LC3-I and -II, were produced post-translationally in various cells. LC3-I is cytosolic, whereas LC3-II is membrane bound. The autophagic vacuole fraction prepared from starved rat liver was enriched with LC3-II. Immunoelectron microscopy on LC3 revealed specific labelling of autophagosome membranes in addition to the cytoplasmic labelling. LC3-II was present both inside and outside of autophagosomes. Mutational analyses suggest that LC3-I is formed by the removal of the C-terminal 22 amino acids from newly synthesized LC3, followed by the conversion of a fraction of LC3-I into LC3-II. The amount of LC3-II is correlated with the extent of autophagosome formation. LC3-II is the first mammalian protein identified that specifically associates with autophagosome membranes.
Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical
and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced
apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of
mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning
confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria.
Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670–675
nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin
A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization
and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical
in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.
Increased proteolysis contributes to muscle atrophy that prevails in many diseases. Elucidating the signalling pathways responsible for this activation is of obvious clinical importance. Autophagy is a ubiquitous degradation process, induced by amino acid starvation, that delivers cytoplasmic components to lysosomes. Starvation markedly stimulates autophagy in myotubes, and the present studies investigate the mechanisms of this regulation. In C(2)C(12) myotubes incubated with serum growth factors, amino acid starvation stimulated autophagic proteolysis independently of p38 and p42/p44 mitogen-activated protein kinases, but in a PI3K (phosphoinositide 3-kinase)-dependent manner. Starvation, however, did not alter activities of class I and class II PI3Ks, and was not sufficient to affect major signalling proteins downstream from class I PI3K (glycogen synthase kinase, Akt/protein kinase B and protein S6). In contrast, starvation increased class III PI3K activity in whole-myotube extracts. In fact, this increase was most pronounced for a population of class III PI3K that coimmunoprecipitated with Beclin1/Apg6 protein, a major determinant in the initiation of autophagy. Stimulation of proteolysis was reproduced by feeding myotubes with synthetic dipalmitoyl-PtdIns3 P, the class III PI3K product. Conversely, protein transfection of anti-class III PI3K inhibitory antibody into starved myotubes inverted the induction of proteolysis. Therefore, independently of class I PI3K/Akt, protein S6 and mitogen-activated protein kinase pathways, amino acid starvation stimulates proteolysis in myotubes by regulating class III PI3K-Beclin1 autophagic complexes.
Macroautophagy mediates the bulk degradation of cytoplasmic components. It accounts for the degradation of most long-lived proteins: cytoplasmic constituents, including organelles, are sequestered into autophagosomes, which subsequently fuse with lysosomes, where degradation occurs. Although the possible involvement of autophagy in homeostasis, development, cell death, and pathogenesis has been repeatedly pointed out, systematic in vivo analysis has not been performed in mammals, mainly because of a limitation of monitoring methods. To understand where and when autophagy occurs in vivo, we have generated transgenic mice systemically expressing GFP fused to LC3, which is a mammalian homologue of yeast Atg8 (Aut7/Apg8) and serves as a marker protein for autophagosomes. Fluorescence microscopic analyses revealed that autophagy is differently induced by nutrient starvation in most tissues. In some tissues, autophagy even occurs actively without starvation treatments. Our results suggest that the regulation of autophagy is organ dependent and the role of autophagy is not restricted to the starvation response. This transgenic mouse model is a useful tool to study mammalian autophagy.
The absence of the outer mitochondrial membrane protein Uth1p was found to induce resistance to rapamycin treatment and starvation,
two conditions that induce the autophagic process. Biochemical studies showed the onset of a fully active autophagic activity
both in wild-type and Δuth1 strains. On the other hand, the disorganization of the mitochondrial network induced by rapamycin treatment or 15 h of nitrogen
starvation was followed in cells expressing mitochondria-targeted green fluorescent protein; a rapid colocalization of green
fluorescent protein fluorescence with vacuole-selective FM4-64 labeling was observed in the wild-type but not in the Δuth1 strain. Degradation of mitochondrial proteins, followed by Western blot analysis, did not occur in mutant strains carrying
null mutations of the vacuolar protease Pep4p, the autophagy-specific protein Atg5p, and Uth1p. These data show that, although
the autophagic machinery was fully functional in the absence of Uth1p, this protein is involved in the autophagic degradation
of mitochondria.
The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases.
The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochondrial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodaminedextran for lysosomes, rhodamine 123 and tetramethylrhodamine methylester (TMRM) for mitochondrial membrane potential (ΓΨ) and propidium iodide for loss of cell viability. During the first 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, numerous surface blebs formed and cell volume increased, but ΓΨ decreased relatively little. Subsequently, the nonspecific permeability of mitochondrial membranes increased, and mitochondria depolarized. These events were followed a few minutes later by disintegration of individual lysosomes. After a few more minutes, viability was lost as indicated by bleb rupture, gross plasma membrane permeability to calcein, and nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine triphosphate (ATP) depletion, bleb formation with cellular swelling, onset of a mitochondrial permeability transition, disintegration of lysosomes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into account this unique sequence of events.
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The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochon-drial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodamine-dextran for lysosomes, rhodamine 123 and tetramethylrhodamine methylester (TMRM) for mitochondrial membrane potential (Δ Ψ) and propidium iodide for loss of cell viability. During the first 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, numerous surface blebs formed and cell volume increased, but Δ Ψ decreased relatively little. Subsequently, the nonspecific permeability of mitochondrial membranes increased, and mitochondria depolarized. These events were followed a few minutes later by disintegration of individual lysosomes. After a few more minutes, viability was lost as indicated by bleb rupture, gross plasma membrane permeability to calcein, and nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine triphosphate (ATP) depletion, bleb formation with cellular swelling, onset of a mitochondrial permeability transition, disintegration of lysosomes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into account this unique sequence of events.
Alcohol is the most abused substance worldwide and a significant source of liver injury; the mechanisms of alcohol-induced liver disease are not fully understood. Significant cellular toxicity and impairment of protein synthesis and degradation occur in alcohol-exposed liver cells, along with changes in energy balance and modified responses to pathogens. Autophagy is the process of cellular catabolism through the lysosomal-dependent machinery, which maintains a balance among protein synthesis, degradation, and recycling of self. Autophagy is part of normal homeostasis and it can be triggered by multiple factors that threaten cell integrity, including starvation, toxins, or pathogens. Multiple factors regulate autophagy; survival and preservation of cellular integrity at the expense of inadequately folded proteins and damaged high-energy generating intracellular organelles are prominent targets of autophagy in pathological conditions. Coincidentally, inadequately folded proteins accumulate and high-energy generating intracellular organelles, such as mitochondria, are damaged by alcohol abuse; these alcohol-induced pathological findings prompted investigation of the role of autophagy in the pathogenesis of alcohol-induced liver damage. Our review summarizes the current knowledge about the role and implications of autophagy in alcohol-induced liver disease.
Using confocal microscopy, onset of the mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by the redistribution of the cytosolic fluorophore, calcein, into mitochondria. Simultaneously, mitochondria release membrane potential-indicating fluorophores like tetramethylrhodamine methylester. The MPT occurs in several forms of necrotic cell death, including oxidative stress, pH-dependent ischemia/reperfusion injury and Ca2+ ionophore toxicity. Cyclosporin A (CsA) and trifluoperazine block the MPT in these models and prevent cell killing, showing that the MPT is a causative factor in necrotic cell death. During oxidative injury induced by t-butylhydroperoxide, onset of the MPT is preceded by pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and an increase of mitochondrial free Ca2+, all changes that promote the MPT. During tissue ischemia, acidosis develops. Because of acidotic pH, anoxic cell death is substantially delayed. However, when pH is restored to normal after reperfusion (reoxygenation at pH 7.4), cell death occurs rapidly (pH paradox). This killing is caused by pH-dependent onset of the MPT, which is blocked by reperfusion at acidotic pH or with CsA. In isolated mitochondria, toxicants causing Reye’s syndrome, such as salicylate and valproate, induce the MPT. Similarly, salicylate induces a CsA-sensitive MPT and killing of cultured hepatocytes. These in vitro findings suggest that the MPT is the pathophysiological mechanism underlying Reye’s syndrome in vivo. Kroemer and coworkers proposed that the MPT is a critical event in the progression of apoptotic cell death. Using confocal microscopy, the MPT can be directly documented during tumor necrosis factor-α induced apoptosis in hepatocytes. CsA blocks this MPT and prevents apoptosis. The MPT does not occur uniformly during apoptosis. Initially, a small proportion of mitochondria undergo the MPT, which increases to nearly 100% over 1–3 h. A technique based on fluorescence resonance energy transfer can selectively reveal mitochondrial depolarization. After nutrient deprivation, a small fraction of mitochondria spontaneously depolarize and enter an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. A model is proposed in which onset of the MPT to increasing numbers of mitochondria within a cell leads progressively to autophagy, apoptosis and necrotic cell death.
Fasting in vivo and nutrient deprivation in vitro enhance sequestration of mitochondria and other organelles by autophagy for recycling of essential nutrients. Here our goal was to use a transgenic mouse strain expressing green fluorescent protein (GFP) fused to rat microtubule-associated protein-1 light chain 3 (LC3), a marker protein for autophagy, to characterize the dynamics of mitochondrial turnover by autophagy (mitophagy) in hepatocytes during nutrient deprivation. In complete growth medium, GFP-LC3 fluorescence was distributed diffusely in the cytosol and incorporated in mostly small (0.2-0.3 μm) patches in proximity to mitochondria, which likely represent preautophagic structures (PAS). After nutrient deprivation plus 1 μM glucagon to simulate fasting, PAS grew into green cups (phagophores) and then rings (autophagosomes) that enveloped individual mitochondria, a process that was blocked by 3-methyladenine. Autophagic sequestration of mitochondria took place in 6.5 ± 0.4 min and often occurred coordinately with mitochondrial fission. After ring formation and apparent sequestration, mitochondria depolarized in 11.8 ± 1.4 min, as indicated by loss of tetramethylrhodamine methylester fluorescence. After ring formation, LysoTracker Red uptake, a marker of acidification, occurred gradually, becoming fully evident at 9.9 ± 1.9 min of ring formation. After acidification, GFP-LC3 fluorescence dispersed. PicoGreen labeling of mitochondrial DNA (mtDNA) showed that mtDNA was also sequestered and degraded in autophagosomes. Overall, the results indicate that PAS serve as nucleation sites for mitophagy in hepatocytes during nutrient deprivation. After autophagosome formation, mitochondrial depolarization and vesicular acidification occur, and mitochondrial contents, including mtDNA, are degraded.
Mitochondrial genomes with deleterious mutations can replicate in cells along with wild-type genomes in a state of heteroplasmy, and are a cause of severe inherited syndromes, such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke (MELAS), neuropathy, ataxia, retinitis pigmentosa-maternally inherited Leigh syndrome (NARP-MILS), and Leber's hereditary optic neuropathy (LHON). The cytosolic E3 ligase, Parkin, commonly mutated in recessive familial parkinsonism, translocates to depolarized mitochondria and induces their autophagic elimination, suggesting that Parkin may signal the selective removal of defective mitochondria within the cell. We report that long-term overexpression of Parkin can eliminate mitochondria with deleterious COXI mutations in heteroplasmic cybrid cells, thereby enriching cells for wild-type mtDNA and restoring cytochrome c oxidase activity. After relieving cybrid cells of Parkin overexpression, a more favorable wild-type to mutant mitochondrial genome ratio is stably maintained. These data support the model that Parkin functions in a mitochondrial quality control pathway. Additionally, they suggest that transiently increasing levels of Parkin expression might ameliorate certain mitochondrial diseases.
Autophagy is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B-II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over-expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure 'autophagic flux' has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals.
To examine the feasibility of optical monitoring of cellular energy states with tissue-transparent near-infrared (NIB) light, the absorption and fluorescence characteristics of Rhodamine 800 in isolated rat liver mitochondria and hepatocytes were investigated. When the dye was incubated with isolated mitochondria, a large red shift of the absorption spectra and quenching of the fluorescence intensity were observed. The absorbance difference at 730 minus 685 nm, or at 730 minus 800 nm, and the fluorescence intensity measured at 692 nm varied linearly with the mitochondrial membrane potential. The spectral changes observed could be explained in terms of the potential-dependent uptake of the dye from the buffer solution into the mitochondrial matrix.The respiration control ratio and oxygen consumption rate were not affected by the addition of Rhodamine 800 at concentrations lower than 5 μM, which was the concentration range mostly employed throughout the present study. In a suspension of hepatocytes, the red shift and fluorescence quenching of Rhodamine 800 characteristic of energized mitochondria were also observed, and these changed to those of the buffer solution with the addition of an uncoupler under normoxia. At the early stage of anoxia, within about 5 min, when cytochrome oxidase was completely reduced, hepatocytes were concluded to be in the fully energized state, since the optical characteristics of Rhodamine 800 were the same as those of energized mitochondria. On the basis of these in vitro data, Rhodamine 800 is concluded to be a possible NIR-active contrast agent, that can be used to monitor the energy states of living tissues, in addition to the tissue oxygenation states, by the use of near-infrared spectrophotometry (NTRS) without harmful effects.
Mitochondrial quality control is important in maintaining proper cellular homeostasis. Although selective mitochondrial degradation by autophagy (mitophagy) is suggested to have an important role in quality control, and though there is evidence for a direct relation between mitophagy and neurodegenerative diseases, the molecular mechanism of mitophagy is poorly understood. Using a screen for mitophagy-deficient mutants, we found that YIL146C/ECM37 is essential for mitophagy. This gene is not required for other types of selective autophagy or for nonspecific macroautophagy. We designated this autophagy-related (ATG) gene as ATG32. The Atg32 protein localizes on mitochondria. Following the induction of mitophagy, Atg32 binds Atg11, an adaptor protein for selective types of autophagy, and is then recruited to and imported into the vacuole along with mitochondria. Therefore, Atg32 confers selectivity for mitochondrial sequestration as a cargo and is necessary for recruitment of this organelle by the autophagy machinery for mitophagy.
Mitochondria are essential organelles that produce most of the energy for a cell, but concomitantly accumulate oxidative damage. Degradation of damaged mitochondria is critical for cell homeostasis, and this process is thought to be mediated by mitophagy, an autophagy-related pathway specific for mitochondria. However, whether mitochondria are selectively degraded, and how the autophagic machinery is targeted to mitochondria, remain largely unknown. Here we demonstrate that, in post-log phase cells under respiratory conditions, a substantial fraction of mitochondria are exclusively sequestered as cargoes and transported to the vacuole, a lytic compartment in yeast, in an autophagy-dependent manner. Interestingly, we found Atg32, a mitochondria-anchored protein essential for mitophagy that is induced during respiratory growth. In addition, our data suggest that Atg32 interacts with Atg8 and Atg11, autophagy-related proteins critical for recognition of cargo receptors. We propose that Atg32 acts as a mitophagy-specific receptor and regulates selective degradation of mitochondria.
Amino acid deprivation and glucagon are both potent inducers of autography and proteolysis in liver. Because glucagon enhanced the metabolic utilization of some amino acids, the catabolic response to both of these stimuli could be achieved by a lowering of intracellular amino acid pools. Alternatively, glucagon could act independently of amino acids. To clarify the mode of hormonal action and also the relationship between the two cellular responses, livers from fed rats were perfused, with and without glucagon, with plasma amino acids over a concentration range of 0 to 10 times normal. Individual amino acids constancy at each level was ensured by perfusion in the single-pass mode. Amino acids alone strongly regulated autophagy and proteolysis in a coordinated fashion; maximal suppression was achieved at twice normal concentration; both effects increased rapidly to maximum at less than normal concentration. Corresponding effects of glucagon, however, could be elicited only at intermediate amino acid levels. None was noted at 4 and 10 times normal; at 0, hormonal stimulation was minimal. The amino acid inhibition was selective because it did not block cyclic AMP production or glycogenolysis. Intracellular pool measurements and systematic alteration of perfusate amino acid composition indicated that the autophagic and proteolytic effects of glucagon are mediated by a hormonally induced depletion of glycine, alanine, glutamate, and glutamine; of these, glutamine alone is the most effective. We conclude that the stimulation of intracellular protein degradation in liver is a manifestation of deprivation-induced autophagy which results from a decrease in certain intracellular glucogenic amino acids, notably glutamine.
The effect of treating mitochondria with visible light above 400 nm on electron transport and coupled reactions was examined. The temporal sequence of changes was: stimulation of respiration coupled to ATP synthesis, a decline in ATP synthesis, inactivation of respiration, increased ATPase activity and, later, loss of the membrane potential. Loss of respiration was principally due to inactivation of dehydrogenases. Of the components of dehydrogenase systems, flavins and quinones were most susceptible to illumination, the iron-sulfur centers were remarkably resistant to being damaged. Succinate dehydrogenase was inactivated before choline and NADH dehydrogenase. Redox reactions of cytochromes and cytochrome c oxidase activity were unaffected. Inactivation was O2-dependent and prevented by anaerobiosis or the presence of substrates for the dehydrogenases. Light in the range 400-500 nm was most effective and the presence of free flavins greatly enhanced inactivation of all of the above mitochondrial activities. This suggests that visible light mediates a flavin-photosensitized reaction that initiates damage involving participation of an activated species of oxygen in the damage propagation.
3-Methyladenine (5 mM) inhibits endogenous protein degradation in isolated rat hepatocytes by about 60%, while having no adverse effect on the degradation of an exogenous protein (asialofetuin), on protein synthesis, or on intracellular ATP levels. 3-Methyladenine appears to act specifically upon the autophagic/lysosomal pathway of degradation, as judged from its lack of effect in the presence of amino acids or a lysosomotropic amine (propylamine). The effect of the purine is not mediated by amino acids because the inhibition of protein degradation is accompanied by a significant depression of intracellular amino acid levels. The ability of 3-methyladenine to suppress the formation of electron microscopically visible autophagosomes suggests that it may be regarded as a specific inhibitor of autophagy.
The methylotrophic yeasts Pichia pastoris and Hansenula polymorpha respond to a methanol substrate by synthesizing peroxisomal enzymes resulting in the formation of large peroxisomes. When the carbon source was changed from methanol to glucose, we observed a rapid loss of peroxisomes. In this comparative study, we utilized biochemical and morphological techniques to characterize the loss of peroxisomes in these yeasts. We used metabolic labeling and chase procedures to evaluate whether this loss was due to suppressed synthesis or enhanced degradation. The synthesis of alcohol oxidase was depressed 10-fold when cultures grown in methanol attained stationary growth. However, no further reduction of synthesis was observed upon transfer of these cultures to glucose medium. In stationary phase cultures maintained in methanol, two peroxisomal proteins, alcohol oxidase and dihydroxyacetone synthase, were degraded with a half-life of over 3 h. However, within 3 h of glucose repression, as much as 80% of the radiolabeled peroxisomal proteins were lost from both yeasts. This glucose-mediated degradative event appeared to be specific for peroxisomal proteins, since mitochondrial proteins were stable. Ultrastructural examination of both yeasts revealed that glucose induced the sequestration of peroxisomes into the yeast vacuole, the presumed site of degradation. These results suggest that peroxisome loss during glucose repression is due to a selective, enhanced degradation of whole peroxisomes by autophagic mechanisms.
To gain an understanding of the mechanisms of lipofuscin (LF) accumulation in aging cells we studied both the formation and the elimination of autophagic vacuoles (AV) in hepatocytes of adult (5-6 months) and old (20-21 months) CBA male mice. For the evaluation of AV formation we determined the degree of AV accumulation 4 h after the injection of vinblastine, which blocks the fusion of AV with lysosomes. Other animals were treated with Triton X-100, which provokes the appearance of AV, and then, after 4 h, with cycloheximide to block the appearance of newly formed AV. AV elimination was then evaluated during the next 30 minutes. In our quantitative electron microscopic study of the liver tissue of these mice, we found a decrease in AV formation rate as well as a decrease in the intensity of AV elimination in hepatocytes of old versus young adult animals. The results indicate that the slowing down of AV elimination with age may play an important role in LF accumulation. Further, LF accumulation manifests itself despite decreased autophagy in aging.
As a first step towards isolation of autophagic sequestering membranes (phagophores), we have purified autophagosomes from rat hepatocytes. Lysosomes were selectively destroyed by osmotic rupture, achieved by incubation of hepatocyte homogenates with the cathepsin C substrate glycyl-phenylalanyl-naphthylamide (GPN). Mitochondria and peroxisomes were removed by Nycodenz gradient centrifugation, and cytosol, microsomes and other organelles by rate sedimentation through metrizamide cushions. The purified autophagosomes were bordered by dual or multiple concentric membranes, suggesting that autophagic sequestration might be performed either by single autophagic cisternae or by cisternal stacks. Okadaic acid, a protein phosphatase inhibitor, disrupted the hepatocytic cytokeratin network and inhibited autophagy completely in intact hepatocytes, perhaps suggesting that autophagy might be dependent on intact intermediate filaments. Vinblastine and cytochalasin D, which specifically disrupted microtubules and microfilaments, respectively, had relatively little (25-30%) inhibitory effect on autophagic sequestration. In a cryo-ultrastructural study, the various autophagic-lysosomal vacuoles were immunogold-labelled, using the cytosolic enzyme superoxide dismutase as an autophagic marker, Lgp120 as a lysosomal membrane marker, and bovine serum albumin as an endocytic marker. Vinblastine (50 microM) was found to inhibit both autophagic and endocytic flux into the lysosomes, with a consequent reduction in lysosomal size. Asparagine (20 mM) caused swelling of the lysosomes, probably as a result of the ammonia formation that could be observed at this high asparagine concentration. Autophagosomes and amphisomes (autophagic-endocytic, prelysosomal vacuoles) accumulated in asparagine-treated cells, reflecting an inhibition of autophagic flux that might be a consequence of lysosomal dysfunction.
Recent studies indicate that phosphatidylinositol 3-kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3-kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3-kinase inhibitors, wortmannin and 2-(4-morpholinyl)-8-phe-nylchromone (LY294002).
The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50, 30 nM) and LY294002 (IC50 10 μM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids.
3-Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3-kinase, thus providing a target for its action.
It is proposed that phosphatidylinositol 3-kinase activity is required for autophagy. 3-Methyladenine inhibits autophagy by inhibition of this enzyme.
Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence. Mitochondrial membrane potential, ROS, onset of MPT, and cell death were monitored with tetramethylrhodamine methyl ester (TMRM), dichlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and the second slower phase occurred during mitochondrial ROS generation. Subsequently, MPT took place, mitochondria depolarized, and cells died. beta-Hydroxybutyrate, which reduces mitochondrial NAD+, delayed cell killing, but lactate, which reduces cytosolic NAD+, did not. Trifluoperazine, which inhibits MPT, did not block the initial oxidation of NAD(P)H but prevented the second phase of oxidation, partially blocked ROS formation, and preserved cell viability. The antioxidants, deferoxamine and diphenylphenylenediamine, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completely and stopped cell killing. Both antioxidants also prevented the mitochondrial permeability transition and subsequent mitochondrial depolarization. In conclusion, NAD(P)H oxidation and ROS formation are critical events promoting MPT in oxidative injury and death of hepatocytes.
To simulate ischemia and reperfusion, cultured rat hepatocytes were incubated in anoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. During anoxia, intracellular pH (pHi) decreased to 6.3, mitochondria depolarized, and ATP decreased to < 1% of basal values, but the mitochondrial permeability transition (MPT) did not occur as assessed by confocal microscopy from the redistribution of cytosolic calcein into mitochondria. Moreover, cell viability remained > 90%. After reperfusion at pH 7.4, pHi returned to pH 7.2, the MPT occurred, and most hepatocytes lost viability. In contrast, after reperfusion at pH 6.2 or with Na(+)-free buffer at pH 7.4, pHi did not rise and cell viability remained > 80%. After acidotic reperfusion, the MPT did not occur. When hepatocytes were reperfused with cyclosporin A (0.5-1 microM) at pH 7.4, the MPT was prevented and cell viability remained > 80%, although pHi increased to 7.2. Reperfusion with glycine (5 mM) also prevented cell killing but did not block recovery of pHi or the MPT. Retention of cell viability was associated with recovery of 30-40% of ATP. In conclusion, preventing the rise of pHi after reperfusion blocked the MPT, improved ATP recovery, and prevented cell death. Cyclosporin A also prevented cell killing by blocking the MPT without blocking recovery of pHi. Glycine prevented cell killing but did not inhibit recovery of pHi or the MPT.
Opening of a high-conductance pore conducting solutes of molecular mass <1,500 Da causes onset of the mitochondrial permeability transition (MPT). Cyclosporin A blocks this pore and prevents acute necrotic cell death in several models. Confocal microscopy directly visualizes onset of the MPT during acute cytotoxicity from the movement of the green-fluorescing fluorophore, calcein, into the mitochondria from the cytosol. The MPT also plays a causative role in tumor necrosis factor-alpha-induced apoptosis in hepatocytes. Progression to apoptosis or necrosis after the MPT may depend on the presence or absence, respectively, of ATP. Often, features of both apoptotic and necrotic cell death develop after death signals and toxic stresses. The term "necrapoptosis" is introduced to emphasize the shared pathways leading to both forms of cell death.
A23187 and related Ca2+ ionophores are widely used to study Ca2+-dependent cell injury. Here, using laser scanning confocal microscopy and parameter-indicating fluorophores, we investigated the role of the mitochondrial permeability transition (MPT) in Br-A23187 toxicity to cultured rat hepatocytes. After 10 microM Br-A23187, over 60% of hepatocytes lost viability within 1 h. This necrotic cell killing was preceded by increased mitochondrial free Ca2+, mitochondrial depolarization, and onset of the MPT. Cyclosporin A (CsA), a blocker of the permeability transition pore, prevented the MPT and cell killing but had no effect on increased mitochondrial free Ca2+ and depolarization after Br-A23187. To determine whether Br-A23187-induced cell killing was linked to loss of cellular ATP supply, hepatocytes were incubated with fructose and oligomycin, a source of glycolytic ATP and an inhibitor of the uncoupler-stimulated mitochondrial ATPase, respectively. Fructose plus oligomycin prevented cell killing after Br-A23187 but not the MPT. When fructose plus oligomycin prevented necrotic cell killing, apoptosis developed after 10 h. When cells were treated additionally with CsA, these apoptotic changes were prevented. In conclusion, the MPT mediates Br-A23187 cytotoxicity. Acutely, the MPT causes mitochondrial uncoupling and profound ATP depletion, which leads to necrotic cell death. However, when glycolytic ATP generation is available, the MPT induces apoptosis. CsA blocks the MPT and prevents both necrotic and apoptotic cell killing after Br-A23187.
Pan caspase inhibitors are potentially powerful cell-protective agents that block apoptosis in response to a wide variety of insults that cause tissue degeneration. In many conditions, however, the blockade of apoptosis by caspase inhibitors does not permit long-term cell survival, but the reasons are not entirely clear. Here we show that the blockade of apoptosis by Boc.Aspartyl(O-methyl)CH2F can result in the highly selective elimination of the entire cohort of mitochondria, including mitochondrial DNA, from both neurons and HeLa cells, irrespective of the stimulus used to trigger apoptosis. In cells that lose their mitochondria, the nuclear DNA, Golgi apparatus, endoplasmic reticulum, centrioles, and plasma membrane remain undamaged. The capacity to remove mitochondria is both specific and regulated since mitochondrial loss in neurons is completely prevented by the expression of the antiapoptotic protein Bcl-2 and partially suppressed by the autolysosomal inhibitor bafilomycin. Cells without mitochondria are more tolerant to an anaerobic environment but are essentially irreversibly committed to death. Prevention of mitochondrial loss may be crucial for the long-term regeneration of tissues emerging from an apoptotic episode in which death was prevented by caspase blockade.
The ubiquitin-proteasome pathway has emerged as a central player in the regulation of several diverse cellular processes. Here, we describe the important components of this complex biochemical machinery as well as several important cellular substrates targeted by this pathway and examples of human diseases resulting from defects in various components of the ubiquitin-proteasome pathway. In addition, this review covers the chemistry of synthetic and natural proteasome inhibitors, emphasizing their mode of actions toward the 20S proteasome. Given the importance of proteasome-mediated protein degradation in various intracellular processes, inhibitors of this pathway will continue to serve as both molecular probes of major cellular networks as well as potential therapeutic agents for various human diseases.
Cells degrade excess and effete organelles by the process of autophagy. Autophagic stimulation of rat hepatocytes by serum deprivation and glucagon (1 M) caused a fivefold increase of spontaneously depolarizing mitochondria to about 1.5% of total mitochondria after 90 min. Cyclosporin A (CsA, 5 M), an immunosuppressant that blocks the mitochondrial permeability transition (MPT), prevented this depolarization. Depolarized mitochondria moved into acidic vacuoles labeled by LysoTracker Red. These autophagosomes also increased several-fold after autophagic stimulation. CsA blocked autophagosomal proliferation, whereas tacrolimus, an immunosuppressant that does not block the MPT, did not. In conclusion, the MPT initiates mitochondrial depolarization after autophagic stimulation and the subsequent sequestration of mitochondria into autophagosomes.
Laser irradiation-induced phototoxicity has been intensively applied in clinical photodynamic therapy for the treatment of a variety of tumors. However, the precise laser damage sites as well as the underlying mechanisms at the subcellular level are unknown. Using a mitochondrial fluorescent marker, MitoTracker Green, severe mitochondrial swelling was noted in laser-irradiated rat brain astrocytes. Nucleus condensation and fragmentation revealed by propidium iodide nucleic acid staining indicated that laser-irradiated cells died from apoptosis. Using an intracellular reactive oxygen species (ROS) fluorescent dye, 2',7'-dichlorofluorescin diacetate, heterogeneous distribution of ROS inside astrocytes was observed after laser irradiation. The level of ROS in the mitochondrial compartment was found to be higher than in other parts of the cell. With another ROS fluorescent dye, dihydrorhodamine-123, and time-lapse laser scanning confocal microscopy, a substantial increase in mitochondrial ROS (mROS) was visualized in visible laser-irradiated astrocytes. The antioxidants melatonin and vitamin E largely attenuated laser irradiation-induced mROS formation and prevented apoptosis. Cyclosporin A (CsA), a mitochondrial permeability transition (MPT) blocker, did not prevent visible laser irradiation-induced mROS formation and apoptosis. In conclusion, mROS formation contributes significantly to visible laser irradiation-induced apoptosis via an MPT-independent pathway.
Low power laser irradiation is regarded to have a significant role in triggering cellular proliferation and in treating diseases of diverse etiologies. The present work contributes to the understanding of the mechanisms of action by studying low power laser effects in human fibroblasts. Confocal laser scanning microscopy is used for irradiation and observation of the same area of interest allowing the imaging of laser effects at the single cell level and in real time. Coverslip cultures were placed in a small incubation chamber for in vivo microscopic observation. Laser stimulation of the cells was performed using the 647 nm line of the confocal laser through the objective lens of the microscope. Mitochondrial membrane potential (ΔΨm), intracellular pH, calcium alterations and generation of reactive oxygen species (ROS) were monitored using specific fluorescent vital probes. The induced effects were quantified using
digital image processing techniques. After laser irradiation, a gradual alkalinization of the cytosolic pH and an increase in mitochondrial membrane potential were observed. Recurrent spikes of intracellular calcium concentration were also triggered by laser. Reactive oxygen species were generated as a result of biostimulation. No such effects were monitored in microscopic fields other than the irradiated ones.
Degradation and turnover of peroxisomes is reviewed. First, we describe the historical aspects of peroxisome degradation research and the two major concepts for breakdown of peroxisomes, i.e., autophagy and autolysis. Next, the comprehensive knowledge on autophagy of peroxisomes in mammalian and yeast cells is reviewed. It has been shown that proliferated peroxisomes are degraded by selective autophagy, and studies using yeast cells have been especially helpful in shedding light on the molecular mechanisms of this process. The degradation of extraperoxisomal urate oxidase crystalloid is noted. Overexpressed wild-type urate oxidase in cultured cells has been shown to be degraded through an unknown proteolytic pathway distinct from the lysosomal system including autophagy or the ubiquitin-proteasome system. Finally, peroxisome autolysis mediated by 15-lipoxygenase (15-LOX) is described. 15-LOX is integrated into the peroxisome membrane causing focal membrane disruptions. The content of the peroxisomes is then exposed to cytosol proteases and seems to be digested quickly. In conclusion, the number of peroxisomes appears to be regulated by two selective pathways, autophagy, including macro- and microautophagy, and 15-LOX-mediated autolysis.
Caloric restriction (CR) and a reduced growth hormone (GH)-insulin-like growth factor (IGF-1) axis are associated with an extension of lifespan across taxa. Evidence is reviewed showing that CR and reduced insulin of GH-IGF-1 axis may exhibit their effects at least partly by their common stimulatory action on autophagy, the cell repair mechanism responsible for the housekeeping of cell membranes and organelles including the free radical generators peroxisomes and mitochondria. It is shown that the life-long weekly administration of an anti-lipolytic drug may decrease glucose and insulin levels and stimulate autophagy and intensify anti-ageing effects of submaximal CR.
The hallmark of eukaryotic cells is compartmentalization of distinct cellular functions into specific organelles. This necessitates the cells to run energetically costly mechanisms to precisely control maintenance and function of these compartments. One of these continuously controls organelle activity and abundance, a process termed homeostasis. Yeast peroxisomes are favorable model systems for studies of organelle homeostasis because both the proliferation and degradation of these organelles can be readily manipulated. Here, we highlight recent achievements in regulation of peroxisome turnover in yeast, in particular Hansenula polymorpha, with a focus on directions of future research.
Autophagy is the major cellular pathway for the degradation of long-lived proteins and cytoplasmic organelles. It involves the rearrangement of subcellular membranes to sequester cargo for delivery to the lysosome where the sequestered material is degraded and recycled. For many decades, it has been known that autophagy occurs in a wide range of eukaryotic organisms and in multiple different cell types during starvation, cellular and tissue remodeling, and cell death. However, until recently, the functions of autophagy in normal development were largely unknown. The identification of a set of evolutionarily conserved genes that are essential for autophagy has opened up new frontiers for deciphering the role of autophagy in diverse biological processes. In this review, we summarize our current knowledge about the molecular machinery of autophagy and the role of the autophagic machinery in eukaryotic development.
Autophagy is the bulk degradation of proteins and organelles, a process essential for cellular maintenance, cell viability, differentiation and development in mammals. Autophagy has significant associations with neurodegenerative diseases, cardiomyopathies, cancer, programmed cell death, and bacterial and viral infections. During autophagy, a cup-shaped structure, the preautophagosome, engulfs cytosolic components, including organelles, and closes, forming an autophagosome, which subsequently fuses with a lysosome, leading to the proteolytic degradation of internal components of the autophagosome by lysosomal lytic enzymes. During the formation of mammalian autophagosomes, two ubiquitylation-like modifications are required, Atg12-conjugation and LC3-modification. LC3 is an autophagosomal ortholog of yeast Atg8. A lipidated form of LC3, LC3-II, has been shown to be an autophagosomal marker in mammals, and has been used to study autophagy in neurodegenerative and neuromuscular diseases, tumorigenesis, and bacterial and viral infections. The other Atg8 homologues, GABARAP and GATE-16, are also modified by the same mechanism. In non-starved rats, the tissue distribution of LC3-II differs from those of the lipidated forms of GABARAP and GATE-16, GABARAP-II and GATE-16-II, suggesting that there is a functional divergence among these three modified proteins. Delipidation of LC3-II and GABARAP-II is mediated by hAtg4B. We review the molecular mechanism of LC3-modification, the crosstalk between LC3-modification and mammalian Atg12-conjugation, and the cycle of LC3-lipidation and delipidation mediated by hAtg4B, as well as recent findings concerning the other two Atg8 homologues, GABARAP and GATE-16. We also highlight recent findings regarding the pathobiology of LC3-modification, including its role in microbial infection, cancer and neuromuscular diseases.
Different mechanisms lead to the degradation of intracellular proteins in the lysosomal compartment. Activation of one autophagic pathway or another, under specific cellular conditions, plays an important role in the ability of the cell to adapt to environmental changes. Each form of autophagy has its own individual characteristics, but it also shares common steps and components with the others. This interdependence of the autophagic pathways confers to the lysosomal system, both specificity and flexibility on substrate degradation. We describe in this review some of the recent findings on the molecular basis and regulation for each of the different autophagic pathways. We also discuss the cellular consequences of their interdependent function. Malfunctioning of the autophagic systems has dramatic consequences, especially in non-dividing differentiated cells. Using the heart as an example of such cells, we analyze the relevance of autophagy in aging and cell death, as well as in different pathological conditions.
In autophagy, portions of cytoplasm are sequestered into autophagosomes and delivered to lysosomes for degradation. Long assumed to be a random process, increasing evidence suggests that autophagy of mitochondria, peroxisomes, and possibly other organelles is selective. A recent paper (Kissova et al., J. Biol. Chem. 2004;279:39068-39074) shows in yeast that a specific outer membrane protein, Uth1p, is required for efficient mitochondrial autophagy. For this selective autophagy of mitochondria, we propose the term "mitophagy" to emphasize the non-random nature of the process. Mitophagy may play a key role in retarding accumulation of somatic mutations of mtDNA with aging.