The mitochondrial UPR: mechanisms, physiological functions and implications in ageing

Key Points

• The mitochondrial unfolded protein response (UPR^mt) is a conserved transcriptional response activated by multiple forms of mitochondrial dysfunction and regulated by mitochondrial-to-nuclear communication.

• The UPR^mt promotes cell survival and mitochondrial recovery through metabolic adaptations and a precise mitochondrial biogenesis programme.

• UPR^mt activation promotes lifespan extension and protects against bacterial pathogens that perturb mitochondrial function.

• Prolonged UPR^mt activation can lead to the propagation of deleterious mitochondrial genomes and mitochondrial damage and can contribute to age-associated organismal deterioration.

• UPR^mt manipulations are potential therapeutic targets for treating the vast number of diseases associated with mitochondrial dysfunction.

Abstract

Mitochondrial function declines during ageing owing to the accumulation of deleterious mitochondrial genomes and damage resulting from the localized generation of reactive oxygen species, both of which are often exacerbated in diseases such as Parkinson disease. Cells have several mechanisms to assess mitochondrial function and activate a transcriptional response known as the mitochondrial unfolded protein response (UPR^mt) when mitochondrial integrity and function are impaired. The UPR^mt promotes cell survival and the recovery of the mitochondrial network to ensure optimal cellular function. Recent insights into the regulation, mechanisms and functions of the UPR^mt have uncovered important and complex links to ageing and ageing-associated diseases. In this Review, we discuss the signal transduction mechanisms that regulate the UPR^mt and the physiological consequences of its activation that affect cellular and organismal health during ageing.

Main

Mitochondria are membrane-bound organelles that are derived from the engulfment of an α-proteobacterium by a pre-eukaryotic cell more than 1 billion years ago^1,2. Like their bacterial ancestor, mitochondria have an outer membrane and an inner membrane that separates the intermembrane space (IMS) from the matrix. Mitochondria harbour small circular genomes (mtDNA) that encode 13 oxidative phosphorylation (OXPHOS) proteins as well as tRNAs and ribosomal RNAs required for their synthesis, which occurs in the mitochondrial matrix³. The remainder of the ∼1,200 mitochondrial proteins are encoded by nuclear genes, synthesized on cytosolic ribosomes and subsequently targeted to and imported into mitochondria⁴.

Mitochondria are best known for being the cellular powerhouses that generate the majority of cellular energy by coupling nutrient oxidation to ATP synthesis via the respiratory chain and ATP synthase. In addition to energy production, mitochondria have other essential metabolic roles, including in amino acid and nucleotide synthesis, iron–sulfur cluster biogenesis, intermediate metabolite biogenesis and calcium homeostasis. Furthermore, mitochondria can function as signalling platforms to regulate programmed cell death and innate immunity⁵.

Mitochondrial and OXPHOS dysfunction is the primary cause of a group of heterogenous disorders known as mitochondrial diseases that most commonly impair neuron or muscle cell function and are typically caused by mutations in genes that encode OXPHOS proteins or genes required for the assembly of the respiratory chain and ATP synthase complexes⁶. Mitochondrial function also declines during ageing owing to the accumulation of damaged mtDNA, increased reactive oxygen species (ROS) generation, toxin exposure and general organelle deterioration. Mitochondrial dysfunction is thought to contribute to the decline in cellular function observed during ageing and to loss of muscle strength and neuronal function^7,8,9,10,11. Age-associated mitochondrial dysfunction is often exacerbated or accelerated in neurodegenerative diseases such as Parkinson disease and Alzheimer disease^7,8.

The mitochondrial unfolded protein response (UPR^mt) is a transcriptional response that is activated by multiple forms of mitochondrial dysfunction and regulated by mitochondrial-to-nuclear communication^{12,13,14,15,16,17,18,19,20}. Cells use a variety of mechanisms to monitor the health of the intracellular mitochondrial network, which is comprised of constantly dividing and fusing organelles to facilitate compartment mixing²¹ and ensure localized mtDNA replication in each mitochondrion²². If mitochondrial function declines, the UPR^mt is activated to promote the repair and recovery of this network and maintain cellular function^13,15,18,23.

A nuanced relationship between ageing and the UPR^mt has become increasingly apparent. For example, the UPR^mt promotes repair and recovery from transient mitochondrial dysfunction caused by toxins or pathogens^15,16,24 and by mutations in nuclear-encoded respiratory chain genes^13,18. However, aberrant or prolonged UPR^mt activation results in cells being in a state of constant mitochondrial recovery that can increase the accumulation of deleterious mitochondrial genomes harbouring loss-of-function point mutations or deletions (ΔmtDNAs) during animal development and ageing that could directly contribute to age-associated deterioration^25,26. Thus, it is important that the UPR^mt is precisely regulated. Recent work has revealed that the UPR^mt is regulated at multiple levels, including at the levels of transcription and chromatin remodelling. Interestingly, chromatin remodelling during mitochondrial dysfunction enables more robust UPR^mt activation in aged cells and promotes longevity^27,28. In addition to intracellular UPR^mt regulation, UPR^mt signalling occurs between cells and tissues, presumably to regulate mitochondrial adaptations throughout an organism^13,29,30,31.

In this Review, we discuss recent insights into the signalling mechanisms that regulate the UPR^mt, including how the integrity of the mitochondrial network is assessed and the changes in transcription and chromatin organization that occur in order to maintain a functional mitochondrial network. We describe the effects of the UPR^mt on cellular and organismal physiology that promote mitochondrial recovery and cell survival during mitochondrial dysfunction and the interactions with other mitochondrial stress responses such as mitophagy. We also discuss the relationship between age-associated mitochondrial deterioration in metazoans and the loss of UPR^mt effectiveness with age^13,32,33.

Activation and regulation of the UPR^mt

The discovery of an adaptive stress response that functions to alleviate the accumulation of unfolded proteins within mitochondria was reported more than 15 years ago in cultured mammalian cells. Overexpression of an irreversibly misfolded mitochondrial protein resulted in increased accumulation of nuclear transcripts encoding several mitochondrial chaperones and proteases, presumably to maintain protein homeostasis (proteostasis) within mitochondria²⁰. As this mitochondrial- specific response is conceptually similar to the transcriptional response activated by protein misfolding in the endoplasmic reticulum known as the UPR, it was named the UPR^mt (Refs 19,34).

Over the past 10 years, genetic and biochemical studies, largely carried out using Caenorhabditis elegans and mammalian model systems, have elucidated the function of the UPR^mt beyond the maintenance of mitochondrial proteostasis, as well as the mechanisms by which mitochondrial dysfunction is detected and communicated to the nucleus to regulate chromatin structure and transcriptional outputs.

A working definition of the UPR^mt. Numerous studies have reported diverse cellular alterations associated with mitochondrial dysfunction. Here, we define the UPR^mt as an ensemble of genes and cellular activities that are transcriptionally regulated by defined transcription factors that are activated following mitochondrial dysfunction. Mitochondrial dysfunction broadly refers to a wide range of defects that include the accumulation of unfolded proteins within the mitochondrial matrix but also conditions that perturb OXPHOS activity, such as genotoxic stress and chemical inhibitors. Below, we discuss recent insights supporting this definition, which continues to rapidly evolve as new discoveries are made.

Mitochondrial network assessment and nuclear communication in C. elegans. Using transcriptional reporters activated by mitochondrial dysfunction, multiple screens in C. elegans have identified factors that are required for UPR^mt activation^{14,16,35,36,37,38}. Consistent with a role in signalling between mitochondria and the nucleus, these factors are localized in mitochondria, the cytosol and the nucleus.

Activating transcription factor associated with stress (ATFS-1) is one of at least two transcription factors required for UPR^mt activation in worms¹⁴. In addition to a nuclear localization sequence (NLS; common to all bZIP transcription factors), ATFS-1 has an amino-terminal mitochondrial-targeting sequence (MTS)¹⁸. This arrangement enables cells to evaluate the function of the mitochondrial network on the basis of mitochondrial protein import efficiency (Fig. 1). In cells with a healthy mitochondrial network, the MTS prevails, and ATFS-1 is imported into mitochondria, where it is degraded by the matrix-localized protease LON. When mitochondria are damaged, the NLS directs ATFS-1 to the nucleus to regulate transcription¹⁸. Importantly, MTS removal or inactivation results in constitutive nuclear accumulation of ATFS-1 and UPR^mt activation^18,39. Thus, compartmentalization of ATFS-1 regulates its transcriptional activity. Moreover, if ATFS-1 fails to be imported into mitochondria, the transcription factor becomes active, indicating that mitochondrial import is a key UPR^mt regulatory event¹⁸.

Figure 1: Perturbations that impair mitochondrial protein import activate the mitochondrial unfolded protein response.

Oxidative phosphorylation (OXPHOS) perturbation, excessive reactive oxygen species (ROS), impaired complex assembly (mitonuclear protein imbalance) and the accumulation of misfolded proteins impair mitochondrial protein import efficiency and activate ATFS-1 (activating transcription factor associated with stress). ATFS-1 is imported into healthy mitochondria via its mitochondrial-targeting sequence (MTS), where it is degraded. If mitochondrial import efficiency is perturbed, ATFS-1 is transported to the nucleus and the mitochondrial unfolded protein response (UPR^mt) is activated. In the nucleus, ATFS-1 induces a number of genes that re-establish mitochondrial function and import efficiency, such as genes that promote recovery of the OXPHOS complexes, re-establish mitochondrial proteostasis by upregulating chaperones and proteases and detoxify ROS, as well as protein import components. NLS, nuclear localization sequence; TIM, translocase of the inner membrane; TOM, translocase of the outer membrane.

PowerPoint slide

Most, if not all, conditions that activate the UPR^mt are also known to influence mitochondrial protein import (Fig. 1; Table 1), which, as indicated by a growing body of evidence, occurs both post-translationally and co-translationally⁴⁰. The majority of proteins targeted to the mitochondrial matrix have an amino-terminal MTS, which first targets them to the translocase of the outer membrane complex (TOM complex) on the mitochondrial outer membrane and then to the translocase of the inner membrane 23 complex (TIM23 complex) in the inner membrane⁴¹. Importantly, protein trafficking through the inner membrane and TIM23 complex requires ATP and the proton gradient generated by the respiratory chain, as well as the matrix-localized chaperone mtHSP70 (also known as HSPA9) and is impaired by high levels of ROS^42,43,44. Various mitochondrial perturbations activate the UPR^mt, including OXPHOS impairment by toxins¹⁶ or by RNAi-mediated knockdown of OXPHOS components and assembly factors^13,45, impaired mitochondrial ribosome function following drug treatment or RNAi-mediated knockdown of genes encoding mitochondrial ribosomal proteins¹⁵ and depletion of mtDNA^17,19 (Table 1). These perturbations induce mitonuclear protein imbalance, whereby the expression of stoichiometric mtDNA-encoded and nuclear-encoded OXPHOS components is mismatched, which results in the accumulation of OXPHOS components unable to assemble into complexes¹⁵. Unassembled OXPHOS components interact with mitochondrial chaperones and must be degraded by localized proteases to prevent toxic effects^46,47. Similarly, impaired mitochondrial protein folding or inhibition of mitochondrial chaperones and proteases^14,18,19 activates the UPR^mt.

Table 1 Defects that activate the mitochondrial unfolded protein response

Owing to the pleiotropic nature of these mitochondrial defects, it is difficult to determine the precise events that lead to UPR^mt activation, but they all impair mitochondrial protein import, consistent with the regulation of ATFS-1 by mitochondrial protein import (Fig. 1). These observations suggest that ATFS-1 is activated by mitochondrial dysfunction that ultimately feeds back on mitochondrial protein import efficiency owing to damage in the system. Consistent with this model, it was recently shown that in Saccharomyces cerevisiae, many mitochondrial proteins mislocalize to the cytosol when mitochondrial proteostasis and protein import are impaired. Interestingly, the mislocalized mitochondrial proteins are extremely toxic if they are not quickly degraded by cytosolic proteasomes^48,49. These data suggest that mitochondrial import is impaired by multiple forms of mitochondrial dysfunction and that multiple pathways are activated in response to such dysfunction.

Mitochondrial network assessment and nuclear communication in mammals. Although the mechanisms of the UPR^mt have been elucidated in C. elegans, the UPR^mt was discovered in mammalian cells with the observation that mitochondrial chaperones and proteases are induced in response to mtDNA depletion or the accumulation of misfolded proteins in the mitochondrial matrix^17,20. More recently, a UPR^mt was found to be induced in mice and cultured mammalian cells by various mitochondrial perturbations, such as deletion of the mitochondrial aspartyl-tRNA synthetase DARS2 (Ref. 50), skeletal-muscle-specific deficiency in CRIF1, which is an integral protein of the large mitochondrial ribosomal subunit (39S)⁵¹, and inhibition of the mitochondrial ribosome, the mitochondrial protease LON and the mitochondrial chaperone tumour necrosis factor type 1 receptor-associated protein (TRAP1; also known as HSP75)^15,52 (Table 1).

Although the resulting transcriptional response to mitochondrial dysfunction is similar in mammals and C. elegans, regulation is probably more complicated. Data obtained over the past 5 years suggest the involvement of three bZIP transcription factors, CHOP (also known as DDIT3), ATF4 and ATF5, associated with the integrated stress response (ISR)^53,54,55,56. The expression of CHOP, ATF4 and ATF5 requires the phosphorylation of the eukaryotic translation initiation factor 2 subunit 1 (eIF2α), which is catalysed by four kinases responsive to diverse cellular stresses (Fig. 2). Some eIF2α molecules are constitutively phosphorylated, but the amount of phosphorylated eIF2α increases when the eIF2α kinases GCN2 and PERK (also known as EIF2AK3) are activated by amino acid depletion and endoplasmic reticulum dysfunction^30,57,58,59, respectively. Increased eIF2α phosphorylation results in reduced global protein synthesis and preferential translation of mRNAs with open reading frames in the 5′ untranslated regions, such as those encoding CHOP, ATF4 and ATF5 (Refs 60,61,62).

Figure 2: Signalling the mammalian mitochondrial unfolded protein response.

Activation of the mammalian mitochondrial unfolded protein response (UPR^mt) requires the phosphorylation of eukaryotic translation initiation factor 2 subunit 1 (eIF2α) by the eIF2α kinases GCN2 and PERK (also known as EIF2AK3), which are activated by amino acid depletion, excessive reactive oxygen species (ROS) or endoplasmic reticulum (ER) stress. Phosphorylation of eIF2α results in reduced protein synthesis and the preferential translation of mRNAs harbouring open reading frames in the 5′ untranslated region, such as the mRNAs encoding the transcription factors CHOP, ATF4 and ATF5. All three seem to be involved in the UPR^mt, although it remains to be determined how their activity is regulated during mitochondrial dysfunction.

PowerPoint slide

Multiple studies have demonstrated that diverse forms of mitochondrial stress induce the expression of CHOP, ATF4 and ATF5 as well as that of genes involved in the UPR^mt, such as mitochondrial proteostasis and metabolic remodelling genes^{30,51,52,63,64,65,66}. For example, mitochondrial chaperones and proteases, as well as CHOP, ATF4 and ATF5, were induced in mice deficient in DARS2 (Ref. 50). Multiple studies have also demonstrated that CHOP, ATF4 or ATF5 are required for the induction of UPR^mt genes during mitochondrial dysfunction, consistent with a model in which these transcription factors regulate the mammalian UPR^mt (Refs 30,51,64,66). Combined, these studies have rediscovered the ISR, but in the context of mitochondrial dysfunction. Almost 15 years ago, ATF4 and PERK were shown to mediate the transcriptional induction of mitochondrial proteostasis genes as well as genes involved in metabolic remodelling⁵³. However, it is not yet clear how each ISR kinase and transcription factor is regulated during mitochondrial dysfunction to achieve a specific mitochondrial stress response, as expression of all three transcription factors can be activated during a variety of conditions, including endoplasmic reticulum stress, amino acid depletion and viral infection, which also increase eIF2α phosphorylation^{60,61,67,68,69,70,71}.

A recent study suggested that ATF5 is a mammalian orthologue of ATFS-1, as ATF5 rescued UPR^mt activation in worms lacking ATFS-1 (Ref. 64). This study also indicated that ATF5 can respond directly to mitochondrial stress because like ATFS-1, ATF5 activity seems to be regulated via mitochondrial import efficiency⁶⁴. During mitochondrial stress, ATF5 was required for the induction of several mitochondrial chaperone and protease genes, consistent with an essential role of ATF5 in cell growth during mitochondrial stress⁶⁴. Interestingly, ATF4 was also shown to protect against mitochondrial stress when cells were depleted of mtDNA⁷² or treated with either paraquat or a mitochondrial protease inhibitor⁶⁶. Protection was mediated by induction of metabolic genes associated with the ISR^3,66 (Table 2).

Table 2 mRNAs induced during the mitochondrial unfolded protein response

It is not clear how the functions of CHOP, ATF4 and ATF5 are coordinated during mitochondrial dysfunction. All three are bZIP proteins, but CHOP and ATF4 do not contain an MTS like ATFS-1 or ATF5 (Ref. 64), suggesting that they are regulated at the level of expression, which requires the induction of eIF2α phosphorylation⁵⁶. One simple hypothesis is that CHOP, ATF4 and eIF2α phosphorylation are sufficient to induce ATF5 expression, consistent with studies indicating that transcription of ATF5 is dependent on CHOP and ATF4 (Refs 54,61,73,74). Once expressed, ATF5 activity can be modulated by mitochondrial function to regulate its transcriptional activity. However, ATF4 and CHOP probably have more direct roles as well.

When perturbations occur within the IMS, activation of the UPR^mt requires the nuclear hormone receptor oestrogen receptor (ERα) in addition to CHOP, ATF4 and ATF5 (Refs 75,76). ERα activation leads to increased proteasome activity to eliminate mislocalized or defective IMS proteins. ERα-mediated signalling also increases expression of the IMS protease HTRA2 and the transcription factor nuclear respiratory factor 1 (NRF1), which promotes mitochondrial biogenesis⁷⁷. Considering the variations in physiology between different mammalian tissues affected by mitochondrial dysfunction, it is possible that other mechanisms of mitochondrial-to-nuclear communication that activate a transcriptional response remain to be discovered.

Chromatin remodelling. Mitochondrial dysfunction stimulates chromatin remodelling required for UPR^mt activation, which also requires mitochondrial-to- nuclear communication. Following mitochondrial stress, the cytosolic protein LIN-65 (which has unknown biochemical function) translocates to nuclei, which requires the cytosol-localized histone lysine methyltransferase MET-2 (homologous to the mammalian protein SETDB1). In the nucleus, LIN-65 and MET-2 promote chromatin compaction and global gene silencing during mitochondrial stress²⁸. Concomitantly, the homeobox transcription factor DVE-1 (homologous to the mammalian DNA-binding proteins SATB1 and SATB2), which is also required for UPR^mt activation³⁵, specifically interacts with chromatin regions harbouring mitochondrial stress response genes to maintain a transcription-competent state and allow UPR^mt activation²⁸ (Fig. 3).

Figure 3: Chromatin remodelling during mitochondrial stress.

Mitochondrial stress leads to chromatin remodelling and global gene silencing. This requires LIN-65 and the incorporation of dimethylated histone H3 at Lys 9 (H3K9me2) in nucleosomes. Histone dimethylation is catalysed by the histone-lysine N-methyltransferase MET-2 in the cytosol . Although unknown (as indicated by the question mark), it is possible that LIN-65 interacts with dimethylated histones in the cytosol to be transported to the nucleus and facilitate histone exchange. Concomitantly, the histone lysine demethylases JMJD-1.2 and JMJD-3.1 promote an open chromatin state by removing methyl groups from H3K27me3, which allows the homeobox transcription factor DVE-1, with its co-activator ubiquitin-like protein 5 (UBL5), to maintain a transcriptional competent state for activating transcription factor associated with stress (ATFS-1) to bind to mitochondrial unfolded protein response elements (UPR^mtE) and activate transcription of UPR^mt genes. MTS, mitochondrial-targeting sequence; NLS, nuclear localization sequence.

PowerPoint slide

MET-2 catalyses histone dimethylation in the cytosol before incorporation into nucleosomes in the nucleus⁷⁸. It is not clear how LIN-65 nuclear accumulation is regulated, but it is possible that LIN-65 interacts with dimethylated histones in the cytosol and traffics, in complex with them, into the nucleus to facilitate histone exchange. Interestingly, nuclear localization of LIN-65 requires the mitochondrial-localized protease CLPP-1 (homologous to the bacterial protease ClpP), which is required for the activation of the UPR^mt in C. elegans³⁵, consistent with LIN-65 receiving mitochondrial-related inputs²⁸.

In addition to a histone methyltransferase, two histone lysine demethylases containing a Jumonji C domain, JMJD-1.2 and JMJD-3.1, are also required for UPR^mt activation. Presumably, the JMJD demethylases promote an open chromatin state at UPR^mt genes to enable DVE-1 and ATFS-1 binding and transcription activation²⁷, whereas LIN-65 and MET-2 mediate global chromatin compaction (Fig. 3). Chromatin remodelling during mitochondrial stress occurs independently of ATFS-1 (Ref. 28). Importantly, in the absence of ATFS-1, LIN-65 accumulates to higher levels in the nucleus, indicating that MET-2 responds to mitochondrial dysfunction via a separate, unknown mechanism²⁸. Notably, the Jumonji methyltransferase activities require both the tricarboxylic acid (TCA) cycle product α-ketoglutarate and iron⁷⁹. As the homeostasis of both molecules is affected by mitochondrial dysfunction, they may contribute to mitochondrial stress stimulation of JMJD-1.2 and JMJD-3.1.

It should be noted that overexpression of JMJD-1.2 is sufficient to activate the UPR^mt, which suggests that it has an upstream role in detecting mitochondrial dysfunction and initiating the response²⁷. However, ATFS-1 mutants lacking the MTS are also able to activate the UPR^mt (Ref. 18), suggesting that the chromatin remodelling machinery and ATFS-1 are stimulated independently and then converge at the relevant UPR^mt genes, where both are required. In conclusion, UPR^mt activation requires at least two inputs responsive to mitochondrial activity. Chromatin remodelling stimulated by coordination of a histone dimethyltransferase and histone demethylases results in nuclear compaction while maintaining the transcriptional activation of UPR^mt genes. In addition, if mitochondrial function is reduced enough to limit import of ATFS-1, the transcription factor traffics to the nucleus to activate the UPR^mt. The temporal regulation of each input remains to be resolved.

UPR^mt regulation via cell-to-cell communication. The UPR^mt activation described above occurs in a cell-autonomous manner. However, mitochondrial dysfunction can lead to UPR^mt activation in neighbouring or distant cells and tissues^13,29,30,31. For example, mitochondrial perturbation in C. elegans neurons results in UPR^mt activation within the neuron but also within intestinal cells, suggesting the presence of a mechanism to coordinate UPR^mt activation between tissues or throughout the organism¹³ (Fig. 4a). Although the mechanisms of signal transduction are still under investigation, it has been proposed that it requires mitokines or other diffusible factors that are released from cells in response to mitochondrial stress to modulate UPR^mt activation in other tissues¹³.

Figure 4: Mitochondrial unfolded protein response in cell–cell communication and innate immunity.

a | Mitochondrial unfolded protein response (UPR^mt) regulation via cell–to–cell communication. Non-cell-autonomous UPR^mt activation is initiated by mitochondrial stress in neurons, which leads to UPR^mt activation in the intestine. Cell–cell communication requires serotonin secretion from serotonergic neurons and the peptide FLP-2, which is secreted from an interneuron that receives environmental inputs. The mechanism by which serotonin and FLP-2 activate the UPR^mt in intestinal cells is unknown, although the receiving intestinal cell requires both activating transcription factor associated with stress (ATFS-1) and chromatin remodelling, which is mediated at least in part by the homeobox transcription factor DVE-1 and its co-activator ubiquitin-like protein 5 (UBL5). b | The role of the UPR^mt in innate immunity. Bacterial pathogens that target and perturb mitochondrial function activate ATFS-1 and the UPR^mt, suggesting that cells monitor mitochondrial function as a means to detect pathogenic or toxic bacteria. ATFS-1 activation in turn results in the induction of antibacterial genes to limit pathogen growth, xenobiotic detoxification genes to eliminate pathogen-derived toxins and a mitochondrial protective response to maintain and promote mitochondrial recovery.

PowerPoint slide

In addition to factors that function in cell-autonomous signalling pathways, non-cell-autonomous UPR^mt activation requires at least two factors that are secreted by neurons. Recently, a neuronal circuit that initiates non-cell-autonomous signalling was identified³¹. Serotonin, secreted from serotonergic neurons, and the peptide FLP-2, secreted from an interneuron that integrates inputs from three sensory neurons, are required for non-cell-autonomous activation (Fig. 4a). However, neither secreted molecule is sufficient to activate the UPR^mt in intestinal cells^29,31, indicating that additional mitokines and their receptors remain to be identified. Importantly, non-cell-autonomous signalling in the receiving intestinal cell requires both ATFS-1 and chromatin remodelling^29,31 (Fig. 4a), raising the interesting question of how neuronal transmission stimulates the chromatin remodelling and transcription activation required for the UPR^mt. Alternative possibilities are that the mitokine either activates each pathway independently of mitochondrial dysfunction or directly alters intestinal mitochondrial function to activate the UPR^mt.

In mammals, the hormones fibroblast growth factor 21 (FGF21) and growth/differentiation factor 15 (GDF15) are induced by multiple forms of mitochondrial dysfunction and have been suggested to be mitokines^{30,50,51,80,81,82,83,84}. FGF21 secretion is induced in an ATF4-dependent manner during mitochondrial stress and promotes β-oxidation and adipose tissue browning of white adipose tissue^30,81. Induction of FGF21, along with mitochondrial chaperones and proteases, was also observed in the hearts of mice lacking DARS2 (Ref. 50). In a skeletal-muscle-specific mitochondria stress model, the induction of GDF15 depends on CHOP and regulates lipolysis and oxidative metabolism in mouse liver and adipocytes⁵¹. Importantly, the levels of FGF21 and GDF15 increase in the serum of patients with mitochondrial diseases and can serve as biomarkers of mitochondrial translation and mtDNA disorders, although FGF21 and GDF15 levels also increase in conditions not obviously related to mitochondrial stress^84,85,86.

Mitochondrial recovery

Mitochondrial dysfunction induces broad changes in gene expression in all organisms examined using transcriptomic^18,66 and proteomic^52,87 approaches. In C. elegans, mitochondrial dysfunction caused by impairment of the mitochondrial quality control protease SPG-7, or the respiratory chain by inhibiting a complex IV component, results in altered expression of over 2,000 transcripts^18,27. Less than half of these require ATFS-1 (Ref. 18); thus, the regulation of only a fraction of the cellular processes affected by UPR^mt activation is understood, which suggests that additional transcription factors are activated during mitochondrial dysfunction⁸⁸. Below, we examine the transcriptional outputs of the UPR^mt mediated by known transcription factors and the interactions with other mitochondrial stress response pathways.

UPR^mt transcriptional outputs. The conditions that activate the UPR^mt also interfere with mitochondrial protein import. For example, OXPHOS perturbation, excessive ROS and mitonuclear protein imbalance (owing to incorrect protein folding or complex assembly) in the matrix or inner membrane all interfere with protein import (Fig. 1). In response, ATFS-1 activates many genes, some of which function to repair these defects (Table 2). ATFS-1 binds to a conserved promoter element (UPR^mtE; very similar to an amino acid response element) in many of these genes required for their induction⁸⁹ (Fig. 3). Of note, ATF4 and ATF5 bind to the same promoter element^64,66,89. To re-establish mitochondrial proteostasis, mitochondrial chaperone and quality control protease genes are induced to facilitate de novo protein folding and degrade those proteins that have become damaged or fail to fold. More specifically related to OXPHOS activity, OXPHOS complex assembly factors, components required for iron–sulfur cluster biogenesis and most mitochondrial ribosome subunits are also induced. In addition, multiple components that limit ROS toxicity, including a superoxide dismutase, glutathione synthesis machinery and ubiquinone synthesis genes, are also induced. Finally, the UPR^mt promotes import recovery by inducing transcription of the core components of the TIM23 complex required to import mitochondrial-targeted proteins into the matrix¹⁸.

UPR^mt activation also results in metabolic remodelling⁸⁹. ATFS-1 temporarily limits OXPHOS gene transcription in both the nucleus and the mitochondria while simultaneously increasing transcription of all glycolysis components^18,89. This shift promotes mitochondrial recovery by temporarily reducing expression of the most highly expressed and difficult to process mitochondrial proteins to limit the protein folding burden on the stressed compartment. Increased glycolysis probably allows cells to generate ATP in the cytosol to promote survival and mitochondrial recovery^89,18. Additionally, loss of the TCA cycle enzyme fumarate hydratase, mitochondrial (FH1; also known as FH), which catalyses the hydration of fumarate to malate, induces ATF4 (Ref. 66) and the UPR^mt in mammals and C. elegans⁹⁰, consistent with metabolic perturbations activating the UPR^mt, which in turn adapts metabolism. Furthermore, mitochondrial dysfunction in flies and mammalian cells was recently found to remodel one-carbon metabolism, which promotes cellular redox balance and nucleotide synthesis^66,72,91. In human cells, mitochondrial dysfunction impaired the serine-dependent synthesis of formate and led to an ATF4-dependent increase in the biosynthesis of serine and transsulfuration in human cells⁷². Importantly, formate or purine supplementation was able to rescue growth and the defects caused by OXPHOS inhibitors, emphasizing the importance of these findings⁷².

Once mitochondrial function and protein import are restored, UPR^mt activity is reduced as ATFS-1 is again efficiently imported into mitochondria and degraded. Thus, because of the short half-life of ATFS-1, the UPR^mt can respond to transient mitochondrial perturbations.

Pathways complementary to the UPR^mt contribute to organelle recovery. To restore mitochondrial proteostasis and organelle recovery, transcriptional adaptations occur at the same time as a reduction in protein synthesis. In both mammals and C. elegans, mitochondrial stress leads to activation of the kinase GCN2, which phosphorylates the translation initiation factor eIF2α to reduce global protein synthesis^45,92 (Fig. 2). This function reduces the influx of newly synthesized proteins into mitochondria so as not to overwhelm the proteostasis machinery and facilitates protein folding and complex assembly⁴⁵. eIF2α phosphorylation in combination with increased transcription mediated by ATFS-1, ATF4 and ATF5 may also prioritize the import of proteins to re-establish proteostasis and adapt metabolism.

A recent study demonstrated that a specific mitochondrial perturbation in C. elegans, caused by inhibition of the mitochondrial chaperone HSP-6 or fatty acid oxidation, led to simultaneous activation of both the UPR^mt and the heat shock response (HSR)⁹³. The activation of the HSR under these conditions was dependent on the activation of the UPR^mt and could reduce proteotoxicity caused by poly-glutamine aggregates⁹³. These findings suggest that the UPR^mt can interact with other stress responses to coordinate the appropriate response to resolve a specific condition⁹³.

The UPR^mt and mitochondrial-specific autophagy (mitophagy), which eliminates severely damaged mitochondria, can be activated simultaneously, as both are responsive to similar forms of mitochondrial dysfunction^25,26,94, including damage by paraquat^38,95, mtDNA mutations or deletions^25,26,96,97 and unfolded protein accumulation in the matrix^20,98. The mitophagy component PTEN-induced putative kinase 1 (PINK1) is imported into healthy mitochondria, processed and targeted for degradation⁹⁹. However, during mitochondrial stress, PINK1 fails to be imported across the inner membrane and instead accumulates on the mitochondrial outer membrane of defective mitochondria^{95,98,100,101}. Here, PINK1 phosphorylates ubiquitin and the ubiquitin ligase parkin, leading to the engulfment of the defective organelle by autophagosomes and subsequent degradation within lysosomes^{95,99,102,103}. The events orchestrated by the UPR^mt and mitophagy are fairly clear (transcription adaptation to promote recovery of mitochondrial activity versus degradation of severely damaged mitochondria); however, how they are coordinated to recover the mitochondrial network remains to be determined.

Implications in physiology and ageing

UPR^mt activation is essential to ensure normal development in the event of mitochondrial stress or OXPHOS dysfunction, consistent with a role of the UPR^mt in inducing metabolic adaptations and a mitochondrial recovery programme^18,45,89. UPR^mt activation is strong during development but relatively mild during adulthood^13,32. However, paradoxically, mild mitochondrial perturbations that activate the UPR^mt during development result in increased longevity in worms, flies and mice^{32,104,105,106,107,108,109,110,111,112}. Conversely, mitochondrial perturbations during adulthood do not extend lifespan or activate the UPR^mt, suggesting that the pathway is differentially regulated as organisms age^13,32,33. It is not clear whether adult cells have reduced capability to detect mitochondrial stress, but it was found that chromatin compaction and remodelling occur near the onset of adulthood and that these chromatin changes limit UPR^mt activation during ageing^27,28,113. Below, we examine physiological roles for UPR^mt signalling during conditions affected by ageing.

The UPR^mt and increased longevity. As stated above, there is a strong correlation between mitochondrial defects that extend lifespan and those that activate the UPR^mt. For example, modest perturbations of OXPHOS complex IV or the mitochondrial ribosome extend lifespan and activate the UPR^mt in worms and mice^13,15,32,114. However, perturbation of OXPHOS complex II (succinate dehydrogenase) activates the UPR^mt but does not extend lifespan in C. elegans^{13,114,115,116}. Multiple studies indicate that the UPR^mt has a role in extending lifespan. For example, inhibition of ATFS-1, the DVE-1 co-activator ubiquitin-like protein 5 (UBL5) and the Jumonji histone demethylases reduces longevity during mitochondrial stress^13,15,27,117. Furthermore, overexpression of the histone demethylases is sufficient to activate the UPR^mt and extend lifespan²⁷. Mitochondrial dysfunction or overexpression of JMJD-1.2 is sufficient to delay chromatin compaction, allowing UPR^mt activation into adulthood²⁷.

Additional mitochondrial stress response pathways are required for increased longevity, consistent with mitochondrial dysfunction being pleiotropic. Hypoxia-inducible factor 1 (HIF-1) is required for lifespan extension during mitochondrial stress, as are several MAP kinases in C. elegans^88,118,119. Mitochondrial-generated ROS also increase longevity in a manner dependent on the programmed cell death machinery in C. elegans¹¹². Although several signalling components that promote longevity following mitochondrial dysfunction have been identified, the cellular processes and physiological changes that are involved remain to be defined. Is metabolic remodelling and/or the activation of genes that limit damage during ageing the cause of increased longevity¹²⁰? Although the longevity-promoting cellular processes affected by the UPR^mt are unclear, it is known that they require chromatin remodelling induced by the demethylases JMJD-1.2 and/or JMJD-1.3, which maintain a chromatin state necessary for UPR^mt activation²⁷.

The UPR^mt in physiology and disease. The UPR^mt seems to be associated with diverse pathologies, which is consistent with mitochondrial dysfunction being common to many diseases. For example, increased levels of transcripts associated with UPR^mt activation have been observed in patients with myopathy caused by mitochondrial disorders⁸⁵, in patients with either sporadic or familial Alzheimer disease¹²¹, in a mouse model of cardiomyopathy caused by loss of a mitochondrial tRNA synthetase⁵⁰, in a mouse model of depression caused by chronic restraint¹²² and in glioblastomas¹²³. Although these observations are consistent with the hypothesis that the UPR^mt becomes activated, the transcription factors required for the observed mitochondrial-stress-induced transcription remain to be determined in each pathological scenario, as do the effects of the transcriptional response on physiology.

Recent evidence suggests that UPR^mt transcripts can function as biomarkers of mitochondrial dysfunction or diseases caused by mitochondrial dysfunction and provide potential therapeutic approaches to treat such conditions. For example, the hormones FGF21 (Ref. 85) and GDF15 (Ref. 51) are biomarkers of myopathy caused by mitochondrial dysfunction. Treatments that increase NAD⁺ activate the UPR^mt and ameliorate mitochondrial function in aged tissues to promote muscle stem cell maintenance and regeneration^124,125. Moreover, administration of GDF15 improved insulin sensitivity in obese mice¹², and supplementation with an NAD⁺ precursor alleviated non-alcoholic fatty liver disease¹²⁶, although it remains to be determined if these improvements are due to UPR^mt activation.

Although the UPR^mt is a stress response, low levels of constitutive UPR^mt activation have been observed and potentially contribute to mitochondrial maintenance during normal development. For example, a recent comparative study of 40 genetically diverse mouse strains revealed a coordinated expression pattern of conserved UPR^mt components, including mitochondrial chaperones and proteases, that negatively correlated with expression of the mitochondrial quality control protease SPG7 in both mice and humans⁸⁷. These data are consistent with SPG-7 inhibition strongly activating the UPR^mt in C. elegans^18,19. These studies suggest that the UPR^mt buffers against more subtle forms of mitochondrial stress that occur in normal populations simply due to genotypic variance. Additionally, mice lacking ATF4 (Ref. 127) or ATF5 (Refs 128,129) have numerous developmental defects consistent with the UPR^mt having baseline functions during normal development.

The UPR^mt as an innate immune response. Both adaptive and innate immunity decline with age and rely on mitochondria for proper regulation^130,131. Mitochondria are signalling platforms for innate immune pathways that are responsive to bacterial and viral infections and produce ROS to eliminate intracellular bacteria¹³⁰. Mitochondria are not only immune response hubs but also targets of pathogen virulence factors¹³². Interestingly, mitochondrial perturbation results in cellular stress responses tightly associated with innate immunity (Fig. 4b). For example, herpesvirus infection results in the release of mtDNA into the cytosol, which activates an antiviral response^130,133. In addition, the bacterium Helicobacter pylori produces a pore-forming protein targeted to mitochondria that perturbs OXPHOS¹³⁴. Similarly, Pseudomonas aeruginosa secretes the OXPHOS inhibitor cyanide and iron-chelating proteins as virulence factors¹³⁵. These observations led to the intriguing hypothesis that monitoring mitochondrial function enables cells to detect pathogens, as mitochondria seem to be common targets of virulence factors¹³⁶.

Recent studies reported that exposure to multiple bacterial species or bacterial-derived toxins activates the UPR^mt (Refs 16,24). Importantly, in addition to a mitochondrial protective transcriptional programme, ATFS-1 induces transcription of multiple antibacterial genes, including antimicrobial peptides and secreted lysozymes, as well as a xenobiotic detoxification programme, suggesting a role in pathogen detection and resistance to toxin exposure^16,24 (Fig. 4b). Exposure to P. aeruginosa activates the UPR^mt, which requires P. aeruginosa to produce cyanide and iron chelators, suggesting that monitoring of mitochondrial function is used to activate an innate immune response. Furthermore, animals lacking ATFS-1 are susceptible to P. aeruginosa accumulation in the intestine, resulting in earlier death. However, hyperactivation of the UPR^mt provides resistance to the pathogen, both slowing intestinal colonization and extending animal survival time, indicating that the UPR^mt is a bona fide innate immune response^16,24,137.

Although the virulence factors that perturb mitochondrial function are largely unknown, a recent survey of natural bacterial isolates revealed that many species cause UPR^mt activation, suggesting that mechanisms to perturb mitochondria are found in many bacterial species and may be widespread¹⁶. It should be noted that the OXPHOS inhibitors antimycin and oligomycin are derived from the bacteria Streptomyces, as is the mitochondrial ribosome inhibitor tetracycline^138,139. While Streptomyces is not a human pathogen, it likely uses these toxins to impair competing bacterial species in the soil where it lives. This suggests that the OXPHOS toxins that evolved to impair competitive bacterial species are also useful virulence factors against mitochondria, which themselves are bacterial-derived.

The UPR^mt and the propagation of deleterious mitochondrial genomes. As discussed above, the decline in UPR^mt activation with ageing potentially contributes to age-associated cell and tissue dysfunction. Thus, approaches to prolong UPR^mt activation during ageing may alleviate age-associated cell dysfunction. However, recent work has indicated that prolonged UPR^mt activation has negative consequences. In addition to reduced fecundity³⁹, prolonged UPR^mt activation promotes, or facilitates, the propagation of deleterious mtDNAs in germ cells and somatic cells, suggesting that the decline in UPR^mt activation during ageing may have been evolutionarily selected^25,26 (Fig. 5a).

Figure 5: Mitochondrial unfolded protein response activation promotes the preferential maintenance and propagation of deleterious mitochondrial genome heteroplasmy.

a | The presence of both wild-type (WT) mitochondrial genomes (mtDNA; blue) and deleterious mutant mtDNAs (ΔmtDNA; red) in the same cell is referred to as heteroplasmy. Accumulation of ΔmtDNA can cause mitochondrial diseases as well as age-associated damage to cells such as muscles and neurons. ΔmtDNA accumulation leads to oxidative phosphorylation dysfunction, which activates activating transcription factor associated with stress (ATFS-1) and the mitochondrial unfolded protein response (UPR^mt). By inducing a mitochondrial recovery programme that includes mitochondrial biogenesis as well as mitochondrial fission and fusion genes, the UPR^mt promotes the preferential propagation of the ΔmtDNA at the expense of the wild-type mtDNA. Severely damaged mitochondria that contain high levels of ΔmtDNA are degraded by mitophagy. b | Inhibition of ATFS-1 and the UPR^mt results in preferential loss of the ΔmtDNA while maintaining the wild-type mtDNA.

PowerPoint slide

Most somatic cells contain hundreds of mtDNAs that accumulate mutations at much higher rates than nuclear genes¹⁴⁰. Mutations or deletions in individual mtDNAs are well tolerated because of the high copy number of mtDNAs in each cell. However, if the percentage of mtDNAs carrying a specific deleterious mutation reaches ∼60–80%, depending on the severity of the loss-of-function mutation and the tissue type, mtDNA mutations can lead to mitochondrial dysfunction, compromising cellular function¹⁴¹. One mechanism to acquire a high load of ΔmtDNAs (mtDNAs carrying deletions) is through inheritance via the maternal germ line. However, deep sequencing data from otherwise healthy individuals have indicated that ΔmtDNAs are commonly found in multiple tissues^141,142. But as the number of mutated mtDNAs is usually quite low, cellular function remains unaffected. However, during ageing, a ΔmtDNA clone can outcompete wild-type mtDNAs, resulting in high levels of heteroplasmy, typically in muscle cells or neurons. The accumulation of ΔmtDNAs during ageing and their damaging effects on cellular function are well-documented and have been reviewed recently¹⁴³. Below, we focus on the interactions between ΔmtDNAs and UPR^mt activation.

The mechanisms that promote the preferential accumulation of ΔmtDNAs in somatic cells are not well understood owing at least in part to the availability of only a few metazoan models that stably inherit easily tractable ΔmtDNAs. However, a heteroplasmic C. elegans strain harbouring ∼60% ΔmtDNAs with a 3-kilobase deletion has provided insight into the maintenance of deleterious heteroplasmies¹⁴⁴. Perhaps not surprisingly, the absence of four essential OXPHOS genes from 60% of the mtDNAs perturbs OXPHOS function and causes UPR^mt activation. UPR^mt activation caused by ΔmtDNAs protects against OXPHOS perturbations in a similar manner to the UPR^mt, protecting cells from mutations in nuclear-encoded OXPHOS genes or toxins that impair OXPHOS¹⁸. Surprisingly, ATFS-1 inhibition caused a near complete loss of the deleterious mtDNA, indicating that UPR^mt activation can preferentially propagate or maintain the deleterious mtDNA (Fig. 5b). Alternatively, further UPR^mt activation resulted in increased accumulation of the ΔmtDNA^25,26. Accumulation of ΔmtDNAs via UPR^mt activation is antagonized by mitophagy, as mitophagy impairment allows further ΔmtDNA accumulation^25,26. Presumably, as the heteroplasmy ratio increases, so does mitochondrial damage, resulting in selective degradation of those mitochondria with the highest ratio of ΔmtDNAs.

One possible mechanism by which the UPR^mt promotes or allows ΔmtDNA accumulation is by mitigating mitochondrial damage, allowing ΔmtDNAs to 'escape' mitophagy. Alternatively, the UPR^mt-mediated mitochondrial recovery programme may more directly promote the propagation of the ΔmtDNA^25,26. Because the UPR^mt responds to a diverse variety of mitochondrial stressors, the repair and recovery programme is not necessarily specific to the underlying cause of mitochondrial stress. For example, exposure to OXPHOS inhibitors elicits a UPR^mt that includes mtDNA replication as part of the recovery programme¹⁸. Similarly, OXPHOS perturbation by ΔmtDNAs also elicits a UPR^mt, but in this case the ΔmtDNA may be preferentially replicated during organelle recovery.

Conclusions and perspectives

New and unexpected roles have been uncovered for the UPR^mt that extend far beyond its original identified role in restoring mitochondrial proteostasis. The rapid rate at which these discoveries have been made suggests that the field will continue to evolve to include additional modes of regulation and physiological roles. Multiple UPR^mt signalling components involved in direct transcriptional adaptations and chromatin remodelling have been found that affect age-associated mitochondrial function. While activation of the UPR^mt is potentially beneficial and may serve as a future therapeutic target, the impact of activating this response long term is not fully understood and should be undertaken cautiously, as prolonged UPR^mt activation can lead to the propagation of mitochondrial damage^25,26,29. Additionally, many of the genes induced by the UPR^mt are of unknown function and should be characterized along with additional transcription factors required to regulate the full transcriptional response to mitochondrial dysfunction^18,88.

The regulation of the UPR^mt in mammals is potentially more complex than that described in C. elegans. In addition, while a mammalian orthologue of ATFS-1 was recently identified⁶⁴, the requirement for at least two additional transcription factors, ATF4 and CHOP, along with four eIF2α kinases suggests that much remains to be determined. For example, which eIF2α kinases respond to mitochondrial dysfunction and how do they do so? The discovery that UPR^mt activation can be transmitted between cells capable of coordinating tissues and protective effects throughout the organism has suggested the existence of mitokines. While the mechanism by which the UPR^mt is communicated is only beginning to be understood, the identification of additional extracellular components will undoubtedly have therapeutic implications as well as contribute to the understanding of the molecular mechanisms that regulate and coordinate the UPR^mt.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.