Mitochondrial Quality Control Mechanisms Research Articles

Insights into mitochondrial quality control pathways and Parkinson’s disease

The brain uses more energy than any other human organ, accounting for 20 % of the body's total demand. Mitochondria are energy-converting organelles with a pivotal role in meeting the energetic needs of the human brain. Therefore, the decline of these cellular powerhouses can have a negative impact on the function and plasticity of neurons and is believed to have a prominent role in ageing and in the occurrence of several neurological disorders, such as Parkinson's disease (PD). As a consequence of their physiological roles, mitochondria are subjected to high levels of stress and have therefore developed several stress-protective mitochondrial quality control mechanisms that ensure the optimal activity of their molecular machinery. Here, we review some of the most recent advances in our understanding of the regulation of mitochondrial stress pathways with particular emphasis on how defective mitochondrial quality control might contribute to PD.

Abstract 1687: Frequent inactivation of the Mieap-regulated mitochondrial quality control in colorectal cancer.

Abstract Recently, we discovered a novel mechanism for mitochondrial quality control (MQC), in which Mieap, a p53-inducible protein, controls MQC by repairing or eliminating unhealthy mitochondria in a process known as MALM (Mieap-induced Accumulation of Lysosome-like organelles within Mitochondria) or MIV (Mieap-Induced Vacuole), respectively (1, 2). We also reported that two mitochondrial outer membrane proteins, BNIP3 and NIX, are essential mediators of the MALM (2, 3). To examine the role of Mieap-regulated MQC pathway in tumorigenesis, we analyzed the status of p53, Mieap, BNIP3, and NIX in 57 cases of primary colorectal cancer. Of the 57 patients, Mieap and BNIP3 promoter methylation was observed in 5 (8.8%) and 26 (45.6%) patients, respectively. A p53 mutation was detected in 4 (18%) of the 22 patients with either Mieap or BNIP3 promoter methylation whereas it was found in 9 (52%) of the 17 patients with neither Mieap nor BNIP3 promoter methylation, implying that the p53/Mieap/BNIP3 pathway in the Mieap-regulated MQC is inactivated in at least 80% of colorectal cancer cases. Hypoxia induced MALM in the colorectal cancer cell line LS174T (wild-type p53, Mieap-positive, BNIP3-positive). In vitro knockdown of p53, Mieap, or BNIP3 in the LS174T cells severely inhibited hypoxia-induced MALM, leading to the accumulation of unhealthy mitochondria and increased mitochondrial reactive oxygen species (mtROS) generation. Furthermore, the mtROS dramatically enhanced cancer migration and invasion under hypoxic conditions. These results suggest that the Mieap-regulated MQC function plays a critical role in tumor suppression in colorectal cancer. 1. Miyamoto Y et al. PLoS ONE 6: e16054, 2011 2. Kitamura N et al. PLoS ONE 6: e16060, 2011 3. Nakamura Y et al. PLoS ONE 7: e30767, 2012 Citation Format: Hiroki Kamino, Yasuyuki Nakamura, Noriaki Kitamura, Manabu Futamura, Masaki Yoshida, Ryuya Murai, Yuri Saito, Hitoya Sano, Yae Kanai, Yoshihiro Moriya, Hirofumi Arakawa. Frequent inactivation of the Mieap-regulated mitochondrial quality control in colorectal cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1687. doi:10.1158/1538-7445.AM2013-1687

Abstract LB-132: Frequent inactivation of the p53/Mieap/BNIP3 mitochondrial quality control pathway in gastric cancer.

Abstract Recently, we discovered a novel mechanism for mitochondrial quality control (MQC), in which Mieap, a p53-inducible protein, controls MQC by repairing or eliminating unhealthy mitochondria via MALM (Mieap-induced Accumulation of Lysosome-like organelles within Mitochondria) or MIV (Mieap-Induced Vacuole) generation, respectively (1, 2). We also reported that two mitochondrial outer membrane proteins, BNIP3 and NIX, are essential mediators of the MALM (2, 3). To examine the role of Mieap-regulated MQC pathway in tumorigenesis, we analyzed the status of p53, Mieap, BNIP3, and NIX in 50 cases of primary gastric cancer. Of the 50 patients, Mieap and BNIP3 promoter methylation was observed in 2 (4%) and 29 (58%) patients, respectively. A p53 mutation was detected in 2 (10%) of the 20 patients with either Mieap or BNIP3 promoter methylation whereas it was found in 3 (15.8%) of the 19 patients with neither Mieap nor BNIP3 promoter methylation, implying that the p53/Mieap/BNIP3 MQC pathway is inactivated in at least 70% of gastric cancer cases. In a colorectal cancer cell line, hypoxia induced MALM. Knockdown of p53, Mieap, or BNIP3 in the cell line severely inhibited hypoxia-induced MALM, leading to the accumulation of unhealthy mitochondria and increased mitochondrial reactive oxygen species (mtROS) generation. Furthermore, the mtROS dramatically enhanced cancer migration and invasion in hypoxic conditions. These results suggest that the p53/Mieap/BNIP3 MQC pathway plays a tumor-suppressive role in gastric cancer. 1. Miyamoto Y et al. PLoS ONE 6: e16054, 2011 2. Kitamura N et al. PLoS ONE 6: e16060, 2011 3. Nakamura Y et al. PLoS ONE 7: e30767, 2012 Citation Format: Hitoya Sano, Hiroki Kamino, Yasuyuki Nakamura, Masaki Yoshida, Ryuya Murai, Yuri Saito, Manabu Futamura, Kazuhiro Yoshida, Hirofumi Arakawa. Frequent inactivation of the p53/Mieap/BNIP3 mitochondrial quality control pathway in gastric cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-132. doi:10.1158/1538-7445.AM2013-LB-132

Oxidative stress and mitochondrial protein quality control in aging

Mitochondrial protein quality control incorporates an elaborate network of chaperones and proteases that survey the organelle for misfolded or unfolded proteins and toxic aggregates. Repair of misfolded or aggregated protein and proteolytic removal of irreversibly damaged proteins are carried out by the mitochondrial protein quality control system. Initial maturation and folding of the nuclear or mitochondrial-encoded mitochondrial proteins are mediated by processing peptidases and chaperones that interact with the protein translocation machinery. Mitochondrial proteins are subjected to cumulative oxidative damage. Thus, impairment of quality control processes may cause mitochondrial dysfunction. Aging has been associated with a marked decline in the effectiveness of mitochondrial protein quality control. Here, we present an overview of the chaperones and proteases involved in the initial folding and maturation of new, incoming precursor molecules, and the subsequent repair and removal of oxidized aggregated proteins. In addition, we highlight the link between mitochondrial protein quality control mechanisms and the aging process. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

The pathways of mitophagy for quality control and clearance of mitochondria.

Selective autophagy of mitochondria, known as mitophagy, is an important mitochondrial quality control mechanism that eliminates damaged mitochondria. Mitophagy also mediates removal of mitochondria from developing erythrocytes, and contributes to maternal inheritance of mitochondrial DNA through the elimination of sperm-derived mitochondria. Recent studies have identified specific regulators of mitophagy that ensure selective sequestration of mitochondria as cargo. In yeast, the mitochondrial outer membrane protein autophagy-related gene 32 (ATG32) recruits the autophagic machinery to mitochondria, while mammalian Nix is required for degradation of erythrocyte mitochondria. The elimination of damaged mitochondria in mammals is mediated by a pathway comprised of PTEN-induced putative protein kinase 1 (PINK1) and the E3 ubiquitin ligase Parkin. PINK1 and Parkin accumulate on damaged mitochondria, promote their segregation from the mitochondrial network, and target these organelles for autophagic degradation in a process that requires Parkin-dependent ubiquitination of mitochondrial proteins. Here we will review recent advances in our understanding of the different pathways of mitophagy. In addition, we will discuss the relevance of these pathways in neurons where defects in mitophagy have been implicated in neurodegeneration.

Nicotinamide-induced Mitophagy: EVENT MEDIATED BY HIGH NAD+/NADH RATIO AND SIRT1 PROTEIN ACTIVATION

Active autophagy coupled with rapid mitochondrial fusion and fission constitutes an important mitochondrial quality control mechanism and is critical to cellular health. In our previous studies, we found that exposure of cells to nicotinamide causes a decrease in mitochondrial content and an increase in mitochondrial membrane potential (MMP) by activating autophagy and inducing mitochondrial fragmentation. Here, we present evidence to show that the effect of nicotinamide is mediated through an increase of the [NAD(+)]/[NADH] ratio and the activation of SIRT1, an NAD(+)-dependent deacetylase that plays a role in autophagy flux. The [NAD(+)]/[NADH] ratio was inversely correlated with the mitochondrial content, and an increase in the ratio by the mobilization of the malate-aspartate shuttle resulted in autophagy activation and mitochondrial transformation from lengthy filaments to short dots. Furthermore, treatment of cells with SIRT1 activators, fisetin or SRT1720, induced similar changes in the mitochondrial content. Importantly, the activators induced mitochondrial fragmentation only when SIRT1 expression was intact. Meanwhile, MMP did not increase when the cells were treated with the activators, suggesting that the change in MMP is not induced by the mitochondrial turnover per se and that elevation of the [NAD(+)]/[NADH] ratio may activate additional mechanisms that cause MMP augmentation. Together, our results indicate that a metabolic state resulting in an elevated [NAD(+)]/[NADH] ratio can modulate mitochondrial quantity and quality via pathways that may include SIRT1-mediated mitochondrial autophagy.

A Vesicular Transport Pathway Shuttles Cargo from Mitochondria to Lysosomes

Mitochondrial respiration relies on electron transport, an essential yet dangerous process in that it leads to the generation of reactive oxygen species (ROS). ROS can be neutralized within the mitochondria through enzymatic activity, yet the mechanism for steady-state removal of oxidized mitochondrial protein complexes and lipids is not well understood. We have previously characterized vesicular profiles budding from the mitochondria that carry selected cargo. At least one population of these mitochondria-derived vesicles (MDVs) targets the peroxisomes; however, the fate of the majority of MDVs was unclear. Here, we demonstrate that MDVs carry selected cargo to the lysosomes. Using a combination of confocal and electron microscopy, we observe MDVs in steady state and demonstrate that they are stimulated as an early response to oxidative stress, the extent of which is determined by the respiratory status of the mitochondria. Delivery to the lysosomes does not require mitochondrial depolarization and is independent of ATG5 and LC3, suggesting that vesicle delivery complements mitophagy. Consistent with this, ultrastructural analysis of MDV formation revealed Tom20-positive structures within the vesicles of multivesicular bodies. These data characterize a novel vesicle transport route between the mitochondria and lysosomes, providing insights into the basic mechanisms of mitochondrial quality control.

Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration

Mitochondrial damage and dysfunction are common hallmarks for neurodegenerative disorders, including Alzheimer, Parkinson, Huntington diseases, and the motor neuron disorder amyotrophic lateral sclerosis. Damaged mitochondria pivotally contribute to neurotoxicity and neuronal cell death in these disorders, e.g., due to their inability to provide the high energy requirements for neurons, their generation of reactive oxygen species (ROS), and their induction of mitochondrion-mediated cell death pathways. Therefore, in-depth analyses of the underlying molecular pathways, including cellular mechanisms controlling the maintenance of mitochondrial function, is a prerequisite for a better understanding of neurodegenerative disorders. The yeast Saccharomyces cerevisiae is an established model for deciphering mitochondrial quality control mechanisms and the distinct mitochondrial roles during apoptosis and programmed cell death. Cell death upon expression of various human neurotoxic proteins has been characterized in yeast, revealing neurotoxic protein-specific differences. This review summarizes how mitochondria are affected in these neurotoxic yeast models, and how they are involved in the execution and prevention of cell death. I will discuss to which extent this mimics the situation in other neurotoxic model systems, and how this may contribute to a better understanding of the mitochondrial roles in the human disorders.

News from the powerhouses

The disastrous 2011 Fukushima earthquake and its sequelae have sparked intense public discussions around a set of very fundamental questions such as whether our society should go on to maintain nuclear powerhouses in the future and if yes, which quality control mechanisms would have to be in place to guarantee the highest level of safety, how their efficiency and obedience themselves could reliably be controlled, and finally, at what costs a potential nuclear power phase-out would come. This scenario is quite reminiscent of the situation on the cellular level, where mitochondria, serving the traditional cliche of ‘‘cellular powerhouses’’, work to satisfy the cells’ energy demands by producing ATP through oxidative phosphorylation and glycolysis. This essential organelle function is, however, invariably coupled to the threat of intracellular damage that—when exceeding the capacity of existing molecular control systems—may easily wreak havoc up to killing the cell from within. On the molecular level, the main perpetrators, reactive oxygen species, leak out of the mitochondrial electron transport chain. Once released, this toxic spill then acts by oxidizing membrane lipids and proteins—including, of course, mitochondrial ones—as well as mitochondrial DNA (mtDNA). Importantly, primarily due to its intronand histone-less structure as well as due to insufficient repair mechanisms, the latter is much more susceptible to mutational damage than the genomic DNA of the nucleus. As a result, in addition to occurring at much higher frequencies in comparison to genomic DNA mutations, mtDNA mutations also accumulate in postmitotic cells such as neurons in an agedependent manner, as reflected clinically by the agedependent manifestation of a number of classical neurodegenerative conditions. To prevent potential damage through leakage of toxic reactive oxygen species from the cellular powerhouses resulting in an age-dependent decline in mitochondrial functions, various quality control mechanisms are in place at the cellular level. One of these mechanisms—dynamic changes in mitochondrial morphology through continuous fission and fusion processes—allows to control the level of mutated mtDNA within cells, keeping thresholds of respiration-deficient mitochondria at bay. In addition, protein turnover pathways as well as degradation of entire damaged or dysfunctional mitochondria represent essential protective mechanisms intimately integrated into cellular physiology. These mechanisms, which have only recently begun to be discovered, are currently the subject of intense research efforts. This is reflected by an impressive increase in the number of respective publications: searching the Web of Knowlegede TM database (Thomson Reuters) for publications using ‘‘mitochondria’’ and ‘‘neurodegeneration’’ as general search terms currently produces more than 1,900 hits, with publication numbers having climbed continuously from less than 75 papers in 2000 to more than 250 yearly publications at present. With dysregulated mitochondrial dynamics having appeared as a new theme in neurodegeneration just a few years ago [1], research on mitochondrial quality control mechanisms has since continued to produce surprising, fundamental new insights into the pathogenesis of certain neurodegenerative disorders. While the interested reader is referred to excellent comprehensive reviews on the general role of mitochondria in neurodegeneration [2], with this mini-cluster of reviews we intend to shed a flashlight on selected, most exciting current developments in the field. S. Frank (&) M. Tolnay Division of Neuropathology, Institute of Pathology, Schonbeinstrasse 40, 4031 Basel, Switzerland e-mail: frankst@uhbs.ch

Neurodegeneration as a consequence of failed mitochondrial maintenance

Maintaining the functional integrity of mitochondria is pivotal for cellular survival. It appears that neuronal homeostasis depends on high-fidelity mitochondria, in particular. Consequently, mitochondrial dysfunction is a fundamental problem associated with a significant number of neurological diseases, including Parkinson's disease (PD), Huntington's disease (HD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS) and various peripheral neuropathies, as well as the normal aging process. To ensure optimal mitochondrial function, diverse, evolutionarily conserved mitochondrial quality control mechanisms are in place, including the scavenging of toxic reactive oxygen species (ROS) and degradation of damaged mitochondrial proteins, but also turnover of whole organelles. In this review we will discuss various mitochondria-associated conditions, focusing on the role of protein turnover in mitochondrial maintenance with special emphasis on neurodegenerative disorders.

Triage of oxidation-prone proteins by Sqstm1/p62 within the mitochondria

As the mitochondrion is vulnerable to oxidative stress, cells have evolved several strategies to maintain mitochondrial integrity, including mitochondrial protein quality control mechanisms and autophagic removal of damaged mitochondria. Involvement of an autophagy adaptor, Sqstm1/p62, in the latter process has been recently described. In the present study, we provide evidence that a portion of p62 directly localizes within the mitochondria and supports stable electron transport by forming heterogeneous protein complexes. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of mitochondrial proteins co-purified with p62 revealed that p62 interacts with several oxidation-prone proteins, including a few components of the electron transport chain complexes, as well as multiple chaperone molecules and redox regulatory enzymes. Accordingly, p62-deficient mitochondria exhibited compromised electron transport, and the compromised function was partially restored by in vitro delivery of p62. These results suggest that p62 plays an additional role in maintaining mitochondrial integrity at the vicinity of target machineries through its function in relation to protein quality control.

Cyclophilin D is required for mitochondrial removal by autophagy in cardiac cells

Autophagy is a highly regulated intracellular degradation process by which cells remove cytosolic long-lived proteins and damaged organelles. The mitochondrial permeability transition (MPT) results in mitochondrial depolarization and increased reactive oxygen species production, which can trigger autophagy. Therefore, we hypothesized that the MPT may have a role in signaling autophagy in cardiac cells. Mitochondrial membrane potential was lower in HL-1 cells subjected to starvation compared to cells maintained in full medium. Mitochondrial membrane potential was preserved in starved cells treated with cyclosporin A (CsA), suggesting the MPT pore is associated with starvation-induced depolarization. Starvation-induced autophagy in HL-1 cells, neonatal rat cardiomyocytes and adult mouse cardiomyocytes was inhibited by CsA. Starvation failed to induce autophagy in CypD-deficient murine cardiomyocytes, whereas in myocytes from mice overexpressing CypD the levels of autophagy were enhanced even under fed conditions. Collectively, these results demonstrate a role for CypD and the MPT in the initiation of autophagy. We also analyzed the role of the MPT in the degradation of mitochondria by biochemical analysis and electron microscopy. HL-1 cells subjected to starvation in the presence of CsA had higher levels of mitochondrial proteins (by Western blot), more mitochondria and less autophagosomes (by electron microscopy) than cells starved in the absence of CsA. Our results suggest a physiologic function for CypD and the MPT in the regulation of starvation-induced autophagy. Starvation-induced autophagy regulated by CypD and the MPT may represent a homeostatic mechanism for cellular and mitochondrial quality control.

A pivotal role for PINK1 and autophagy in mitochondrial quality control: implications for Parkinson disease

The PTEN-induced putative kinase 1 (PINK1) is a mitochondrially targeted serine-threonine kinase, which is linked to autosomal recessive familial parkinsonism. Current literature implicates PINK1 as a pivotal regulator of mitochondrial quality control, promoting maintenance of respiring mitochondrial networks through cristae stabilization, phosphorylation of chaperones and possibly regulation of mitochondrial transport or autophagy. Pulse-chase studies indicate that PINK1 is rapidly processed into at least two shorter forms, which are distributed in both mitochondrial and cytosolic compartments. Through indirect regulation of mitochondrial proteases and Drp1, PINK1 may act to facilitate localized repair and fusion in response to minor mitochondrial stress. With severe mitochondrial damage, PINK1 facilitates aggregation and clearance of depolarized mitochondria through interactions with Parkin and possibly Beclin1. This switch in function most probably involves altered processing, post-translational modification and/or localization of PINK1, as overexpression of full-length PINK1 is required for mitochondrial Parkin recruitment. Under conditions of PINK1 deficiency, dysregulation of reactive oxygen species, electron transport chain function and calcium homeostasis trigger altered mitochondrial dynamics, indicating compromise of mitochondrial quality control mechanisms. Nevertheless, Parkin- and Beclin1-regulated mitochondrial autophagy remains effective at recycling PINK1-deficient mitochondria; failure of this final tier of mitochondrial quality control contributes to cell death. Thus, PINK1 plays a pivotal, multifactorial role in mitochondrial homeostasis. As autophagic recycling represents the final tier of mitochondrial quality control, whether PINK1 levels are enhanced or reduced, strategies to promote selective mitophagy and mitochondrial biogenesis may prove effective for multiple forms of Parkinson's disease.

The molecular mechanism of mitochondria autophagy in yeast

Mitochondria are critical for supplying energy to the cell, but during catabolism this organelle also produces reactive oxygen species that can cause oxidative damage. Accordingly, quality control of mitochondria is important to maintain cellular homeostasis. It has been assumed that autophagy is the pathway for mitochondrial recycling, and that the selective degradation of mitochondria via autophagy (mitophagy) is the primary mechanism for mitochondrial quality control, although there is little experimental evidence to support this idea. Recent studies in yeast identified several mitophagy-related genes and have uncovered components involved in the molecular mechanism and regulation of mitophagy. Similarly, studies of Parkinson disease and reticulocyte maturation reveal that Parkin and Nix, respectively, are required for mitophagy in mammalian cells, and these analyses have revealed important physiological roles for mitophagy. Here, we review the current knowledge on mitophagy, in particular on the molecular mechanism and regulation of mitophagy in yeast. We also discuss some of the differences between yeast and mammalian mitophagy.