From discovery of the CHOP axis and targeting ClpP to the identification of additional axes of the UPRmt driven by the estrogen receptor and SIRT3

The mitochondrial UPR (UPR^mt) is rapidly gaining attention. While most studies on the UPR^mt have focused on its role in aging, emerging studies suggest an important role of the UPR^mt in cancer. Further, several of the players of the UPR^mt in mammalian cells have well reported roles in the maintenance of the organelle. The goal of this review is to emphasize aspects of the UPR^mt that have been overlooked in the current literature, describe the role of specific players of the UPR^mt in the biology of the mitochondria and highlight the intriguing possibility that targeting the UPR^mt in cancer may be already within reach.     

Inhibition of the mitochondrial unfolded protein response by acetylcholine alleviated hypoxia/reoxygenation-induced apoptosis of endothelial cells

The mitochondrial unfolded protein response (UPR^mt) is involved in numerous diseases that have the common feature of mitochondrial dysfunction. However, its pathophysiological relevance in the context of hypoxia/reoxygenation (H/R) in endothelial cells remains elusive. Previous studies have demonstrated that acetylcholine (ACh) protects against cardiomyocyte injury by suppressing generation of mitochondrial reactive oxygen species (mtROS). This study aimed to explore the role of UPR^mt in endothelial cells during H/R and to clarify the beneficial effects of ACh. Our results demonstrated that H/R triggered UPR^mt in endothelial cells, as evidenced by the elevation of heat shock protein 60 and LON protease 1 protein levels, and resulted in release of mitochondrial pro-apoptotic proteins, including cytochrome C, Omi/high temperature requirement protein A 2 and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low PI, from the mitochondria to cytosol. ACh administration markedly decreased UPR^mt by inhibiting mtROS and alleviating the mitonuclear protein imbalance. Consequently, ACh alleviated the release of pro-apoptotic proteins and restored mitochondrial ultrastructure and function, thereby reducing the number of terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. Intriguingly, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3AChR) inhibitor, abolished the ACh-elicited attenuation of UPR^mt and TUNEL positive cells, indicating that the salutary effects of ACh were likely mediated by M3AChR in endothelial cells. In conclusion, our studies demonstrated that UPR^mt might be essential for triggering the mitochondrion-associated apoptotic pathway during H/R. ACh markedly suppressed UPR^mt by inhibiting mtROS and alleviating the mitonuclear protein imbalance, presumably through M3AChR.     

Effect of the mitochondrial unfolded protein response on hypoxic death and mitochondrial protein aggregation

Mitochondria are the main oxygen consumers in cells and as such are the primary organelle affected by hypoxia. All hypoxia pathology presumably derives from the initial mitochondrial dysfunction. An early event in hypoxic pathology in C. elegans is disruption of mitochondrial proteostasis with induction of the mitochondrial unfolded protein response (UPR^mt) and mitochondrial protein aggregation. Here in C. elegans, we screen through RNAis and mutants that confer either strong resistance to hypoxic cell death or strong induction of the UPR^mt to determine the relationship between hypoxic cell death, UPR^mt activation, and hypoxia-induced mitochondrial protein aggregation (HIMPA). We find that resistance to hypoxic cell death invariantly mitigated HIMPA. We also find that UPR^mt activation invariantly mitigated HIMPA. However, UPR^mt activation was neither necessary nor sufficient for resistance to hypoxic death and vice versa. We conclude that UPR^mt is not necessarily hypoxia protective against cell death but does protect from mitochondrial protein aggregation, one of the early hypoxic pathologies in C. elegans.Subject terms: Necroptosis, Energy metabolism     

Mitochondrial S-adenosylmethionine deficiency induces mitochondrial unfolded protein response and extends lifespan in Caenorhabditis elegans

S-adenosylmethionine (SAM), generated from methionine and ATP by S-adenosyl methionine synthetase (SAMS), is the universal methyl group donor required for numerous cellular methylation reactions. In Caenorhabditis elegans, silencing sams-1, the major isoform of SAMS, genetically or via dietary restriction induces a robust mitochondrial unfolded protein response (UPR^mt) and lifespan extension. In this study, we found that depleting SAMS-1 markedly decreases mitochondrial SAM levels. Moreover, RNAi knockdown of SLC-25A26, a carrier protein responsible for transporting SAM from the cytoplasm into the mitochondria, significantly lowers the mitochondrial SAM levels and activates UPR^mt, suggesting that the UPR^mt induced by sams-1 mutations might result from disrupted mitochondrial SAM homeostasis. Through a genetic screen, we then identified a putative mitochondrial tRNA methyltransferase TRMT-10C.2 as a major downstream effector of SAMS-1 to regulate UPR^mt and longevity. As disruption of mitochondrial tRNA methylation likely leads to impaired mitochondrial tRNA maturation and consequently reduced mitochondrial translation, our findings suggest that depleting mitochondrial SAM level might trigger UPR^mt via attenuating protein translation in the mitochondria. Together, this study has revealed a potential mechanism by which SAMS-1 regulates UPR^mt and longevity.     

线粒体未折叠蛋白反应分子机制的研究进展

线粒体未折叠蛋白反应(UPR~(mt))作为新发现的细胞内应激机制,直接影响老化、神经退行性疾病、癌症等疾病的发生发展.UPR~(mt)是线粒体为了维持其内部蛋白质的平衡,启动由核DNA编码的线粒体热休克蛋白和蛋白酶等基因群转录活化程序的应激反应.深入探究UPR~(mt)的作用机制对阐明老化和线粒体相关疾病的发病机理具有指导意义.本文主要阐述了线粒体未折叠蛋白反应的诱导因素、线虫和哺乳动物细胞中最新的未折叠蛋白应激反应的信号传导通路、调控因子、具体作用机制以及线粒体未折叠蛋白反应与衰老、免疫等疾病的联系,旨在为这些疾病提供新的理论基础和治疗靶点.     

SirT3 Regulates the Mitochondrial Unfolded Protein Response

The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death. Therefore, mechanisms to overcome mitochondrial stress are needed. The unfolded protein response (UPR) triggered by accumulation of misfolded proteins in the mitochondria (UPR^mt) has been reported recently. So far, the UPR^mt has been reported to involve the activation of CHOP and estrogen receptor alpha (ERα). The current study describes a novel role of the mitochondrial deacetylase SirT3 in the UPR^mt. Our data reveal that SirT3 acts to orchestrate two pathways, the antioxidant machinery and mitophagy. Inhibition of SirT3 in cells undergoing proteotoxic stress severely impairs the mitochondrial network and results in cellular death. These observations suggest that SirT3 acts to sort moderately stressed from irreversibly damaged organelles. Since SirT3 is reported to act as a tumor suppressor during transformation, our findings reveal a dual role of SirT3. This novel role of SirT3 in established tumors represents an essential mechanism of adaptation of cancer cells to proteotoxic and mitochondrial stress.     

Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt

The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPR^mt) in Caenorhabditis elegans. Far less is known about mammalian UPR^mt signaling, although similar roles were assumed for central players, including CLPP. To better understand the mammalian UPR^mt signaling, we deleted CLPP in hearts of DARS2‐deficient animals that show robust induction of UPR^mt due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPR^mt in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPR^mt signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases.     

Prohibitin-Mediated Lifespan and Mitochondrial Stress Implicate SGK-1, Insulin/IGF and mTORC2 in C. elegans

Lifespan regulation by mitochondrial proteins has been well described, however, the mechanism of this regulation is not fully understood. Amongst the mitochondrial proteins profoundly affecting ageing are prohibitins (PHB-1 and PHB-2). Paradoxically, in C. elegans prohibitin depletion shortens the lifespan of wild type animals while dramatically extending that of metabolically compromised animals, such as daf-2-insulin-receptor mutants. Here we show that amongst the three kinases known to act downstream of daf-2, only loss of function of sgk-1 recapitulates the ageing phenotype observed in daf-2 mutants upon prohibitin depletion. Interestingly, signalling through SGK-1 receives input from an additional pathway, parallel to DAF-2, for the prohibitin-mediated lifespan phenotype. We investigated the effect of prohibitin depletion on the mitochondrial unfolded protein response (UPR^mt). Remarkably, the lifespan extension upon prohibitin elimination, of both daf-2 and sgk-1 mutants, is accompanied by suppression of the UPR^mt induced by lack of prohibitin. On the contrary, gain of function of SGK-1 results in further shortening of lifespan and a further increase of the UPR^mt in prohibitin depleted animals. Moreover, SGK-1 interacts with RICT-1 for the regulation of the UPR^mt in a parallel pathway to DAF-2. Interestingly, prohibitin depletion in rict-1 loss of function mutant animals also causes lifespan extension. Finally, we reveal an unprecedented role for mTORC2-SGK-1 in the regulation of mitochodrial homeostasis. Together, these results give further insight into the mechanism of lifespan regulation by mitochondrial function and reveal a cross-talk of mitochondria with two key pathways, Insulin/IGF and mTORC2, for the regulation of ageing and stress response.     

ATF5, a putative therapeutic target for the mitochondrial DNA 3243A > G mutation-related disease

The mitochondrial DNA m.3243A > G mutation is well-known to cause a variety of clinical phenotypes, including diabetes, deafness, and osteoporosis. Here, we report isolation and expansion of urine-derived stem cells (USCs) from patients carrying the m.3243A > G mutation, which demonstrate bimodal heteroplasmy. USCs with high levels of m.3243A > G mutation displayed abnormal mitochondrial morphology and function, as well as elevated ATF5-dependent mitochondrial unfolded protein response (UPR^mt), together with reduced Wnt/β-catenin signaling and osteogenic potentials. Knockdown of ATF5 in mutant USCs suppressed UPR^mt, improved mitochondrial function, restored expression of GSK3B and WNT7B, and rescued osteogenic potentials. These results suggest that ATF5-dependent UPR^mt could be a core disease mechanism underlying mitochondrial dysfunction and osteoporosis related to the m.3243A > G mutation, and therefore could be a novel putative therapeutic target for this genetic disorder.Subject terms: Mechanisms of disease, Diabetes     

Surveillance-Activated Defenses Block the ROS–Induced Mitochondrial Unfolded Protein Response

Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPR^mt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPR^mt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPR^mt. Activation of the UPR^mt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of kgb-1 abrogated the inhibition of ROS–induced UPR^mt, suggesting that surveillance-activated defenses specifically inhibit the UPR^mt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the Drosophila PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism.     

Resistance of glia-like central and peripheral neural stem cells to genetically induced mitochondrial dysfunction—differential effects on neurogenesis

Mitochondria play a central role in stem cell homeostasis. Reversible switching between aerobic and anaerobic metabolism is critical for stem cell quiescence, multipotency, and differentiation, as well as for cell reprogramming. However, the effect of mitochondrial dysfunction on neural stem cell (NSC) function is unstudied. We have generated an animal model with homozygous deletion of the succinate dehydrogenase subunit D gene restricted to cells of glial fibrillary acidic protein lineage (hGFAP-SDHD mouse). Genetic mitochondrial damage did not alter the generation, maintenance, or multipotency of glia-like central NSCs. However, differentiation to neurons and oligodendrocytes (but not to astrocytes) was impaired and, hence, hGFAP-SDHD mice showed extensive brain atrophy. Peripheral neuronal populations were normal in hGFAP-SDHD mice, thus highlighting their non-glial (non hGFAP⁺) lineage. An exception to this was the carotid body, an arterial chemoreceptor organ atrophied in hGFAP-SDHD mice. The carotid body contains glia-like adult stem cells, which, as for brain NSCs, are resistant to genetic mitochondrial damage.     

PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix

Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson''s disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPR^mt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPR^mt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.     

Mitochondrial stress: a bridge between mitochondrial dysfunction and metabolic diseases?

Under pathophysiological conditions such as obesity, excessive oxidation of nutrients may induce mitochondrial stress, leading to mitochondrial unfolded protein response (UPR^mt) and initiation of a retrograde stress signaling pathway. Defects in the UPR^mt and the retrograde signaling pathways may disrupt the integrity and homeostasis of the mitochondria, resulting in endoplasmic reticulum stress and insulin resistance. Improving the capacity of mitochondria to reduce stress may be an effective approach to improve mitochondria function and to suppress obesity-induced metabolic disorders such as insulin resistance and type 2 diabetes.     

Depletion of TMEM65 leads to oxidative stress,apoptosis, induction of mitochondrial unfolded protein response,and upregulation of mitochondrial protein import receptor TOMM22

Mutation in the transmembrane protein 65 gene (TMEM65) results in mitochondrial dysfunction and a severe mitochondrial encephalomyopathy phenotype. However, neither the function of TMEM65 nor the cellular responses to its depletion have been fully elucidated. Hence, we knocked down TMEM65 in human cultured cells and analyzed the resulting cellular responses. Depletion of TMEM65 led to a mild increase in ROS generation and upregulation of the mRNA levels of oxidative stress suppressors, such as NFE2L2 and SESN3, indicating that TMEM65 knockdown induced an oxidative stress response. A mild induction of apoptosis was also observed upon depletion of TMEM65. Depletion of TMEM65 upregulated protein levels of the mitochondrial chaperone HSPD1 and mitochondrial protease LONP1, indicating that mitochondrial unfolded protein response (UPR^mt) was induced in response to TMEM65 depletion. Additionally, we found that the mitochondrial protein import receptor TOMM22 and HSPA9 (mitochondrial Hsp70), were also upregulated in TMEM65-depleted cells. Notably, the depletion of TMEM65 did not lead to upregulation of TOMM22 in an ATF5-dependent manner, although upregulation of LONP1 reportedly occurs in an ATF5-dependent manner. Taken together, our findings suggest that depletion of TMEM65 causes mild oxidative stress and apoptosis, induces UPR^mt, and upregulates protein expression of mitochondrial protein import receptor TOMM22 in an ATF5-independent manner.     

Loss of mitochondrial protease ClpP protects mice from diet‐induced obesity and insulin resistance

Caseinolytic peptidase P (ClpP) is a mammalian quality control protease that is proposed to play an important role in the initiation of the mitochondrial unfolded protein response (UPR^mt), a retrograde signaling response that helps to maintain mitochondrial protein homeostasis. Mitochondrial dysfunction is associated with the development of metabolic disorders, and to understand the effect of a defective UPR^mt on metabolism, ClpP knockout (ClpP^?/?) mice were analyzed. ClpP^?/? mice fed ad libitum have reduced adiposity and paradoxically improved insulin sensitivity. Absence of ClpP increased whole‐body energy expenditure and markers of mitochondrial biogenesis are selectively up‐regulated in the white adipose tissue (WAT) of ClpP^?/? mice. When challenged with a metabolic stress such as high‐fat diet, despite similar caloric intake, ClpP^?/? mice are protected from diet‐induced obesity, glucose intolerance, insulin resistance, and hepatic steatosis. Our results show that absence of ClpP triggers compensatory responses in mice and suggest that ClpP might be dispensable for mammalian UPR^mt initiation. Thus, we made an unexpected finding that deficiency of ClpP in mice is metabolically beneficial.