Host-microbe interactions rewire metabolism in a C. elegans model of leucine breakdown deficiency.

In humans, defects in leucine catabolism cause a variety of inborn errors in metabolism. Here, we use Caenorhabditis elegans to investigate the impact of mutations in mccc-1, an enzyme that functions in leucine breakdown. Through untargeted metabolomic and transcriptomic analyses we find extensive metabolic rewiring that helps to detoxify leucine breakdown intermediates via conversion into previously undescribed metabolites and to synthesize mevalonate, an essential metabolite. We also find that the leucine breakdown product 3,3-hydroxymethylbutyrate (HMB), commonly used as a human muscle-building supplement, is toxic to C. elegans and that bacteria modulate this toxicity. Unbiased genetic screens revealed interactions between the host and microbe, where components of bacterial pyrimidine biosynthesis mitigate HMB toxicity. Finally, upregulated ketone body metabolism genes in mccc-1 mutants provide an alternative route for biosynthesis of the mevalonate precursor 3-hydroxy-3-methylglutaryl-CoA. Our work demonstrates that a complex host-bacteria interplay rewires metabolism to allow host survival when leucine catabolism is perturbed.

Mitochondrial inorganic polyphosphate is required to maintain proteostasis within the organelle.

The existing literature points towards the presence of robust mitochondrial mechanisms aimed at mitigating protein dyshomeostasis within the organelle. However, the precise molecular composition of these mechanisms remains unclear. Our data show that inorganic polyphosphate (polyP), a polymer well-conserved throughout evolution, is a component of these mechanisms. In mammals, mitochondria exhibit a significant abundance of polyP, and both our research and that of others have already highlighted its potent regulatory effect on bioenergetics. Given the intimate connection between energy metabolism and protein homeostasis, the involvement of polyP in proteostasis has also been demonstrated in several organisms. For example, polyP is a bacterial primordial chaperone, and its role in amyloidogenesis has already been established. Here, using mammalian models, our study reveals that the depletion of mitochondrial polyP leads to increased protein aggregation within the organelle, following stress exposure. Furthermore, mitochondrial polyP is able to bind to proteins, and these proteins differ under control and stress conditions. The depletion of mitochondrial polyP significantly affects the proteome under both control and stress conditions, while also exerting regulatory control over gene expression. Our findings suggest that mitochondrial polyP is a previously unrecognized, and potent component of mitochondrial proteostasis.

Itaconate impairs immune control of Plasmodium by enhancing mtDNA-mediated PD-L1 expression in monocyte-derived dendritic cells.

Severe forms of malaria are associated with systemic inflammation and host metabolism disorders; however, the interplay between these outcomes is poorly understood. Using a Plasmodium chabaudi model of malaria, we demonstrate that interferon (IFN) γ boosts glycolysis in splenic monocyte-derived dendritic cells (MODCs), leading to itaconate accumulation and disruption in the TCA cycle. Increased itaconate levels reduce mitochondrial functionality, which associates with organellar nucleic acid release and MODC restraint. We hypothesize that dysfunctional mitochondria release degraded DNA into the cytosol. Once mitochondrial DNA is sensitized, the activation of IRF3 and IRF7 promotes the expression of IFN-stimulated genes and checkpoint markers. Indeed, depletion of the STING-IRF3/IRF7 axis reduces PD-L1 expression, enabling activation of CD8+ T cells that control parasite proliferation. In summary, mitochondrial disruption caused by itaconate in MODCs leads to a suppressive effect in CD8+ T cells, which enhances parasitemia. We provide evidence that ACOD1 and itaconate are potential targets for adjunct antimalarial therapy.

Regulation of proteostasis and innate immunity via mitochondria-nuclear communication.

Mitochondria are perhaps best known as the "powerhouse of the cell" for their role in ATP production required for numerous cellular activities. Mitochondria have emerged as an important signaling organelle. Here, we first focus on signaling pathways mediated by mitochondria-nuclear communication that promote protein homeostasis (proteostasis). We examine the mitochondrial unfolded protein response (UPRmt) in C. elegans, which is regulated by a transcription factor harboring both a mitochondrial- and nuclear-targeting sequence, the integrated stress response in mammals, as well as the regulation of chromatin by mitochondrial metabolites. In the second section, we explore the role of mitochondria-to-nuclear communication in the regulation of innate immunity and inflammation. Perhaps related to their prokaryotic origin, mitochondria harbor molecules also found in viruses and bacteria. If these molecules accumulate in the cytosol, they elicit the same innate immune responses as viral or bacterial infection.

ATFS-1 counteracts mitochondrial DNA damage by promoting repair over transcription.

The ability to balance conflicting functional demands is critical for ensuring organismal survival. The transcription and repair of the mitochondrial genome (mtDNA) requires separate enzymatic activities that can sterically compete1, suggesting a life-long trade-off between these two processes. Here in Caenorhabditis elegans, we find that the bZIP transcription factor ATFS-1/Atf5 (refs. 2,3) regulates this balance in favour of mtDNA repair by localizing to mitochondria and interfering with the assembly of the mitochondrial pre-initiation transcription complex between HMG-5/TFAM and RPOM-1/mtRNAP. ATFS-1-mediated transcriptional inhibition decreases age-dependent mtDNA molecular damage through the DNA glycosylase NTH-1/NTH1, as well as the helicase TWNK-1/TWNK, resulting in an enhancement in the functional longevity of cells and protection against decline in animal behaviour caused by targeted and severe mtDNA damage. Together, our findings reveal that ATFS-1 acts as a molecular focal point for the control of balance between genome expression and maintenance in the mitochondria.

Mitochondrial genome recovery by ATFS-1 is essential for development after starvation.

Nutrient availability regulates the C. elegans life cycle as well as mitochondrial physiology. Food deprivation significantly reduces mitochondrial genome (mtDNA) numbers and leads to aging-related phenotypes. Here we show that the bZIP (basic leucine zipper) protein ATFS-1, a mediator of the mitochondrial unfolded protein response (UPRmt), is required to promote growth and establish a functional germline after prolonged starvation. We find that recovery of mtDNA copy numbers and development after starvation requires mitochondrion-localized ATFS-1 but not its nuclear transcription activity. We also find that the insulin-like receptor DAF-2 functions upstream of ATFS-1 to modulate mtDNA content. We show that reducing DAF-2 activity represses ATFS-1 nuclear function while causing an increase in mtDNA content, partly mediated by mitochondrion-localized ATFS-1. Our data indicate the importance of the UPRmt in recovering mitochondrial mass and suggest that atfs-1-dependent mtDNA replication precedes mitochondrial network expansion after starvation.

The hepatic integrated stress response suppresses the somatotroph axis to control liver damage in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) can be ameliorated by calorie restriction, which leads to the suppressed somatotroph axis. Paradoxically, the suppressed somatotroph axis is associated with patients with NAFLD and is correlated with the severity of fibrosis. How the somatotroph axis becomes dysregulated and whether the repressed somatotroph axis impacts liver damage during the progression of NAFLD are unclear. Here, we identify a regulatory branch of the hepatic integrated stress response (ISR), which represses the somatotroph axis in hepatocytes through ATF3, resulting in enhanced cell survival and reduced cell proliferation. In mouse models of NAFLD, the ISR represses the somatotroph axis, leading to reduced apoptosis and inflammation but decreased hepatocyte proliferation and exacerbated fibrosis in the liver. NAD+ repletion reduces the ISR, rescues the dysregulated somatotroph axis, and alleviates NAFLD. These results establish that the hepatic ISR suppresses the somatotroph axis to control cell fate decisions and liver damage in NAFLD.

Mitochondrial dysfunction, aging, and the mitochondrial unfolded protein response in Caenorhabditis elegans.

We review the findings that establish that perturbations of various aspects of mitochondrial function, including oxidative phosphorylation, can promote lifespan extension, with different types of perturbations acting sometimes independently and additively on extending lifespan. We also review the great variety of processes and mechanisms that together form the mitochondrial unfolded protein response. We then explore the relationships between different types of mitochondrial dysfunction-dependent lifespan extension and the mitochondrial unfolded protein response. We conclude that, although several ways that induce extended lifespan through mitochondrial dysfunction require a functional mitochondrial unfolded protein response, there is no clear indication that activation of the mitochondrial unfolded protein response is sufficient to extend lifespan, despite the fact that the mitochondrial unfolded protein response impacts almost every aspect of mitochondrial function. In fact, in some contexts, mitochondrial unfolded protein response activation is deleterious. To explain this pattern, we hypothesize that, although triggered by mitochondrial dysfunction, the lifespan extension observed might not be the result of a change in mitochondrial function.

Impaired mitochondrial oxidative metabolism in skeletal progenitor cells leads to musculoskeletal disintegration.

Although skeletal progenitors provide a reservoir for bone-forming osteoblasts, the major energy source for their osteogenesis remains unclear. Here, we demonstrate a requirement for mitochondrial oxidative phosphorylation in the osteogenic commitment and differentiation of skeletal progenitors. Deletion of Evolutionarily Conserved Signaling Intermediate in Toll pathways (ECSIT) in skeletal progenitors hinders bone formation and regeneration, resulting in skeletal deformity, defects in the bone marrow niche and spontaneous fractures followed by persistent nonunion. Upon skeletal fracture, Ecsit-deficient skeletal progenitors migrate to adjacent skeletal muscle causing muscle atrophy. These phenotypes are intrinsic to ECSIT function in skeletal progenitors, as little skeletal abnormalities were observed in mice lacking Ecsit in committed osteoprogenitors or mature osteoblasts. Mechanistically, Ecsit deletion in skeletal progenitors impairs mitochondrial complex assembly and mitochondrial oxidative phosphorylation and elevates glycolysis. ECSIT-associated skeletal phenotypes were reversed by in vivo reconstitution with wild-type ECSIT expression, but not a mutant displaying defective mitochondrial localization. Collectively, these findings identify mitochondrial oxidative phosphorylation as the prominent energy-driving force for osteogenesis of skeletal progenitors, governing musculoskeletal integrity.

LONP-1 and ATFS-1 sustain deleterious heteroplasmy by promoting mtDNA replication in dysfunctional mitochondria.

The accumulation of deleterious mitochondrial DNA (∆mtDNA) causes inherited mitochondrial diseases and ageing-associated decline in mitochondrial functions such as oxidative phosphorylation. Following mitochondrial perturbations, the bZIP protein ATFS-1 induces a transcriptional programme to restore mitochondrial function. Paradoxically, ATFS-1 is also required to maintain ∆mtDNAs in heteroplasmic worms. The mechanism by which ATFS-1 promotes ∆mtDNA accumulation relative to wild-type mtDNAs is unclear. Here we show that ATFS-1 accumulates in dysfunctional mitochondria. ATFS-1 is absent in healthy mitochondria owing to degradation by the mtDNA-bound protease LONP-1, which results in the nearly exclusive association between ATFS-1 and ∆mtDNAs in heteroplasmic worms. Moreover, we demonstrate that mitochondrial ATFS-1 promotes the binding of the mtDNA replicative polymerase (POLG) to ∆mtDNAs. Interestingly, inhibition of the mtDNA-bound protease LONP-1 increased ATFS-1 and POLG binding to wild-type mtDNAs. LONP-1 inhibition in Caenorhabditis elegans and human cybrid cells improved the heteroplasmy ratio and restored oxidative phosphorylation. Our findings suggest that ATFS-1 promotes mtDNA replication in dysfunctional mitochondria by promoting POLG-mtDNA binding, which is antagonized by LONP-1.

ATF-4 and hydrogen sulfide signalling mediate longevity in response to inhibition of translation or mTORC1.

Inhibition of the master growth regulator mTORC1 (mechanistic target of rapamycin complex 1) slows ageing across phyla, in part by reducing protein synthesis. Various stresses globally suppress protein synthesis through the integrated stress response (ISR), resulting in preferential translation of the transcription factor ATF-4. Here we show in C. elegans that inhibition of translation or mTORC1 increases ATF-4 expression, and that ATF-4 mediates longevity under these conditions independently of ISR signalling. ATF-4 promotes longevity by activating canonical anti-ageing mechanisms, but also by elevating expression of the transsulfuration enzyme CTH-2 to increase hydrogen sulfide (H2S) production. This H2S boost increases protein persulfidation, a protective modification of redox-reactive cysteines. The ATF-4/CTH-2/H2S pathway also mediates longevity and increased stress resistance from mTORC1 suppression. Increasing H2S levels, or enhancing mechanisms that H2S influences through persulfidation, may represent promising strategies for mobilising therapeutic benefits of the ISR, translation suppression, or mTORC1 inhibition.

Genome-wide detection of CRISPR editing in vivo using GUIDE-tag.

Analysis of off-target editing is an important aspect of the development of safe nuclease-based genome editing therapeutics. in vivo assessment of nuclease off-target activity has primarily been indirect (based on discovery in vitro, in cells or via computational prediction) or through ChIP-based detection of double-strand break (DSB) DNA repair factors, which can be cumbersome. Herein we describe GUIDE-tag, which enables one-step, off-target genome editing analysis in mouse liver and lung. The GUIDE-tag system utilizes tethering between the Cas9 nuclease and the DNA donor to increase the capture rate of nuclease-mediated DSBs and UMI incorporation via Tn5 tagmentation to avoid PCR bias. These components can be delivered as SpyCas9-mSA ribonucleoprotein complexes and biotin-dsDNA donor for in vivo editing analysis. GUIDE-tag enables detection of off-target sites where editing rates are ≥ 0.2%. UDiTaS analysis utilizing the same tagmented genomic DNA detects low frequency translocation events with off-target sites and large deletions in vivo. The SpyCas9-mSA and biotin-dsDNA system provides a method to capture DSB loci in vivo in a variety of tissues with a workflow that is amenable to analysis of gross genomic alterations that are associated with genome editing.

UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import in Caenorhabditis elegans.

As organisms develop, individual cells generate mitochondria to fulfill physiological requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS). Here, using the model organism Caenorhabditis elegans we demonstrate that ATFS-1 mediates an adaptable mitochondrial network expansion program that is active throughout normal development. Mitochondrial network expansion requires the relatively inefficient MTS in ATFS-1, which allows the transcription factor to be responsive to parameters that impact protein import capacity of the mitochondrial network. Increasing the strength of the ATFS-1 MTS impairs UPRmt activity by increasing accumulation within mitochondria. Manipulations of TORC1 activity increase or decrease ATFS-1 activity in a manner that correlates with protein synthesis. Lastly, expression of mitochondrial-targeted GFP is sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation.

Targeting the De Novo Purine Synthesis Pathway Through Adenylosuccinate Lyase Depletion Impairs Liver Cancer Growth by Perturbing Mitochondrial Function.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is among the most common cancer types worldwide, yet patients with HCC have limited treatment options. There is an urgent need to identify drug targets that specifically inhibit the growth of HCC cells. APPROACH AND RESULTS: We used a CRISPR library targeting ~2,000 druggable genes to perform a high-throughput screen and identified adenylosuccinate lyase (ADSL), a key enzyme involved in the de novo purine synthesis pathway, as a potential drug target for HCC. ADSL has been implicated as a potential oncogenic driver in some cancers, but its role in liver cancer progression remains unknown. CRISPR-mediated knockout of ADSL impaired colony formation of liver cancer cells by affecting AMP production. In the absence of ADSL, the growth of liver tumors is retarded in vivo. Mechanistically, we found that ADSL knockout caused S-phase cell cycle arrest not by inducing DNA damage but by impairing mitochondrial function. Using data from patients with HCC, we also revealed that high ADSL expression occurs during tumorigenesis and is linked to poor survival rate. CONCLUSIONS: Our findings uncover the role of ADSL-mediated de novo purine synthesis in fueling mitochondrial ATP production to promote liver cancer cell growth. Targeting ADSL may be a therapeutic approach for patients with HCC.

Mitochondrial translation, dynamics, and lysosomes combine to extend lifespan.

In this issue, Liu et al. (2019. J. Cell. Biol.https://doi.org/10.1083/jcb.201907067) find that the inhibition of mitochondrial ribosomes in combination with impaired mitochondrial fission or fusion increases C. elegans lifespan by activating the transcription factor HLH-30, which promotes lysosomal biogenesis.

Folding the Mitochondrial UPR into the Integrated Stress Response.

Eukaryotic cells must accurately monitor the integrity of the mitochondrial network to overcome environmental insults and respond to physiological cues. The mitochondrial unfolded protein response (UPRmt) is a mitochondrial-to-nuclear signaling pathway that maintains mitochondrial proteostasis, mediates signaling between tissues, and regulates organismal aging. Aberrant UPRmt signaling is associated with a wide spectrum of disorders, including congenital diseases as well as cancers and neurodegenerative diseases. Here, we review recent research into the mechanisms underlying UPRmt signaling in Caenorhabditis elegans and discuss emerging connections between the UPRmt signaling and a translational regulation program called the 'integrated stress response'. Further study of the UPRmt will potentially enable development of new therapeutic strategies for inherited metabolic disorders and diseases of aging.

Histone deacetylases 1 and 2 silence cryptic transcription to promote mitochondrial function during cardiogenesis.

Cryptic transcription occurs widely across the eukaryotic genome; however, its regulation during vertebrate development is not understood. Here, we show that two class I histone deacetylases, Hdac1 and Hdac2, silence cryptic transcription to promote mitochondrial function in developing murine hearts. Mice lacking Hdac1 and Hdac2 in heart exhibit defective developmental switch from anaerobic to mitochondrial oxidative phosphorylation (OXPHOS), severe defects in mitochondrial mass, mitochondrial function, and complete embryonic lethality. Hdac1/Hdac2 promotes the transition to OXPHOS by enforcing transcriptional fidelity of metabolic gene programs. Mechanistically, Hdac1/Hdac2 deacetylates histone residues including H3K23, H3K14, and H4K16 to suppress cryptic transcriptional initiation within the coding regions of actively transcribed metabolic genes. Thus, Hdac1/2-mediated epigenetic silencing of cryptic transcription is essential for mitochondrial function during early vertebrate development.