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Cancers arise through the accumulation of genetic and epigenetic alterations that enable cells to evade telomere-based proliferative barriers and achieve immortality. One such barrier is replicative crisis—an autophagy-dependent program that eliminates checkpoint-deficient cells with unstable telomeres and other cancer-relevant chromosomal aberrations1,2. However, little is known about the molecular events that regulate the onset of this important tumour-suppressive barrier. Here we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as a regulator of the crisis program. A crisis-associated isoform of ZBP1 is induced by the cGAS–STING DNA-sensing pathway, but reaches full activation only when associated with telomeric-repeat-containing RNA (TERRA) transcripts that are synthesized from dysfunctional telomeres. TERRA-bound ZBP1 oligomerizes into filaments on the outer mitochondrial membrane of a subset of mitochondria, where it activates the innate immune adapter protein mitochondrial antiviral-signalling protein (MAVS). We propose that these oligomerization properties of ZBP1 serve as a signal amplification mechanism, where few TERRA–ZBP1 interactions are sufficient to launch a detrimental MAVS-dependent interferon response. Our study reveals a mechanism for telomere-mediated tumour suppression, whereby dysfunctional telomeres activate innate immune responses through mitochondrial TERRA–ZBP1 complexes to eliminate cells destined for neoplastic transformation.

Cells respond to mitochondrial poisons with rapid activation of the adenosine monophosphate–activated protein kinase (AMPK), causing acute metabolic changes through phosphorylation and prolonged adaptation of metabolism through transcriptional effects. Transcription factor EB (TFEB) is a major effector of AMPK that increases expression of lysosome genes in response to energetic stress, but how AMPK activates TFEB remains unresolved. We demonstrate that AMPK directly phosphorylates five conserved serine residues in folliculin-interacting protein 1 (FNIP1), suppressing the function of the folliculin (FLCN)–FNIP1 complex. FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB and TFEB-dependent increases of peroxisome proliferator–activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) messenger RNAs. Thus, mitochondrial damage triggers AMPK-FNIP1–dependent nuclear translocation of TFEB, inducing sequential waves of lysosomal and mitochondrial biogenesis.

According to the endosymbiotic theory, most of the DNA of the original bacterial endosymbiont has been lost or transferred to the nucleus, leaving a much smaller (∼16 kb in mammals), circular molecule that is the present-day mitochondrial DNA (mtDNA). The ability of mtDNA to escape mitochondria and integrate into the nuclear genome was discovered in budding yeast, along with genes that regulate this process. Mitochondria have emerged as key regulators of innate immunity, and it is now recognized that mtDNA released into the cytoplasm, outside of the cell, or into circulation activates multiple innate immune signaling pathways. Here, we first review the mechanisms through which mtDNA is released into the cytoplasm, including several inducible mitochondrial pores and defective mitophagy or autophagy. Next, we cover how the different forms of released mtDNA activate specific innate immune nucleic acid sensors and inflammasomes. Finally, we discuss how intracellular and extracellular mtDNA release, including circulating cell-free mtDNA that promotes systemic inflammation, are implicated in human diseases, bacterial and viral infections, and senescence and aging. Expected final online publication date for the Annual Review of Biochemistry, Volume 92 is June 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Acquired resistance to HER2-targeted therapies occurs frequently in HER2+ breast tumors and new strategies for overcoming resistance are needed. Here, we report that resistance to trastuzumab is reversible, as resistant cells regained sensitivity to the drug after being cultured in drug-free media. RNA-sequencing analysis showed that cells resistant to trastuzumab or trastuzumab + pertuzumab in combination increased expression of oxidative phosphorylation pathway genes. Despite minimal changes in mitochondrial respiration, these cells exhibited increased expression of ATP synthase genes and selective dependency on ATP synthase function. Resistant cells were sensitive to inhibition of ATP synthase by oligomycin A, and knockdown of ATP5J or ATP5B, components of ATP synthase complex, rendered resistant cells responsive to a low dose of trastuzumab. Furthermore, combining ATP synthase inhibitor oligomycin A with trastuzumab led to regression of trastuzumab-resistant tumors in vivo. In conclusion, we identify a novel vulnerability of cells with acquired resistance to HER2-targeted antibody therapies and reveal a new therapeutic strategy to overcome resistance.Significance:These findings implicate ATP synthase as a novel potential target for tumors resistant to HER2-targeted therapies.

Supplementary Table 2: BT-TR1 versus BT474 RNA sequencing normalized gene expression Supplementary Table 3: BT-TR2 versus BT474 RNA sequencing normalized gene expression Supplementary Table 4: BT-TPR1 versus BT474 RNA sequencing normalized gene expression Supplementary Table 5: BT-TPR2 versus BT474 RNA sequencing normalized gene expression Supplementary Table 6: BT-TR1 versus BT474 positively enriched hallmark pathways. Supplementary Table 7: BT-TR2 versus BT474 positively enriched hallmark pathways. Supplementary Table 8: BT-TPR1 versus BT474 positively enriched hallmark pathways. Supplementary Table 9: BT-TPR2 versus BT474 positively enriched hallmark pathways. Supplementary Table 10: BT-TR1 versus BT474 negatively enriched hallmark pathways. Supplementary Table 11: BT-TR2 versus BT474 negatively enriched hallmark pathways. Supplementary Table 12: BT-TPR1 versus BT474 negatively enriched hallmark pathways. Supplementary Table 13: BT-TPR2 versus BT474 negatively enriched hallmark pathways.

Supplementary Figure 1: Generation and characterization of resistant cell pools. Supplementary Figure 2: Copy number assessment of receptor tyrosine kinases in resistant cell pools. Supplementary Figure 3: Trastuzumab resistance is reversible. Supplementary Figure 4: Drug resistant cells are more sensitive to OXPHOS inhibitors than parental cells. Supplementary Figure 5: Drug resistant cells have similar mitochondrial DNA copy numbers and ATP levels as the parental cells. Supplementary Figure 6: Glycolysis in parental BT474 versus trastuzumab resistant cells. Supplementary Figure 7: ATP synthase gene expression in BT474, SKBR3 cells and their derived trastuzumab-resistant cells. Supplementary Figure 8: Cumulative survival of breast cancer (all, basal, luminal) patients with patients divided in half by ATP5B (left) or ATP5A1 (right) mRNA level. Supplementary Figure 9: ATP5B protein expression correlation with survival in HER2+, trastuzumab treated patients from HeCOG 10/05. Supplementary Figure 10: ATP5B is required for the maintenance of resistance. Supplementary Figure 11: BT-TR2 tumor morphology and proliferation. Supplementary Table 1: Sequences of primers and plasmid inserts. Supplementary Methods Supplementary References

High copy number, damage prone, and lean on repair mechanisms are unique features of mitochondrial DNA (mtDNA) that are hard to reconcile with its essentiality for oxidative phosphorylation, the primary function ascribed to this maternally inherited component of our genome. We propose that mtDNA is also a genotoxic stress sentinel, as well as a direct second messenger of this type of cellular stress. Here, we discuss existing evidence for this sentinel/effector role through the ability of mtDNA to escape the confines of the mitochondrial matrix and activate nuclear DNA damage/repair responses via interferon-stimulated gene products and other downstream effectors. However, this arrangement may come at a cost, leading to cancer chemoresistance and contributing to inflammation, disease pathology, and aging.

Maternally inherited mitochondrial DNA (mtDNA) encodes essential subunits of the mitochondrial oxidative phosphorylation system, but is also a major damage-associated molecular pattern (DAMP) that engages innate immune sensors when released into the cytoplasm, outside of cells or into circulation1. This function of mtDNA contributes to antiviral resistance, but unfortunately also causes pathogenic inflammation in many disease contexts2. Cells experiencing mtDNA stress due to depletion of the mtDNA-packaging protein, Transcription Factor A, Mitochondrial (TFAM), or HSV-1 infection exhibit elongated mitochondria, mtDNA depletion, enlargement of nucleoids (mtDNA-protein complexes), and activation of cGAS/STING innate immune signaling via mtDNA released into the cytoplasm3. However, the relationships between altered mitochondrial dynamics and mtDNA-mediated activation of the cGAS-STING pathway remain unclear. Here, we show that entire enlarged nucleoids are released from mitochondria that remain bound to TFAM and colocalize with cGAS. These nucleoids arise at sites of mtDNA replication due to a block in mitochondrial fission at a stage when endoplasmic reticulum (ER) actin polymerization would normally commence, which we propose is a fission checkpoint to ensure that mtDNA has completed replication and is competent for segregation into daughter mitochondria. Released nucleoids also colocalize with the early endosomal marker RAB5 as well as the late endosomal marker RAB7 in TFAM-deficient cells and in response to mtDNA stress caused by the HSV-1 UL12.5 protein. Loss of RAB7 increases interferon stimulated gene (ISG) expression. Thus, we propose that defects in mtDNA replication and/or segregation enact a late mitochondrial fission checkpoint that, if persistent, leads to selective removal of dysfunctional nucleoids by a mitochondrial-endosomal pathway. Early steps in this pathway are prone to mtDNA release and cGAS-STING activation, but the immunostimulatory mtDNA is ultimately disposed of through a mechanism involving RAB7-containing late endosomes to prevent excessive innate immune signaling. This mtDNA quality control pathway might represent a therapeutic target to prevent mtDNA-mediated inflammation and associated pathology.

Senescent cells produce chronic inflammation that contributes to the diseases and debilities of aging. How this process is orchestrated in epithelial cells, the origin of human carcinomas, is poorly understood. We used human normal oral keratinocytes (NOKs) to elucidate senescence programs in a prototype primary mucosal epithelial cell that senesces spontaneously. While NOKs exhibit several typical facets of senescence, they also display distinct characteristics. These include expression of p21WAF1/CIP1 at early passages, making this common marker of senescence unreliable in NOKs. Transcriptome analysis by RNA-seq revealed specific commonalities with and differences from cancer cells, explicating the tumor avoidance role of senescence. Repression of DNA repair genes that correlated with downregulation of E2F1 mRNA and protein was observed for two donors; a divergent result was seen for the third. Using proteomic profiling of soluble (non-vesicular) and extracellular vesicle (EV) associated secretions, we propose additions to the senescence associated secretory phenotype, including HSP60, which localizes to the surface of EVs. Finally, EVs from senescent NOKs activate interferon pathway signaling in THP-1 monocytes in a STING-dependent manner and associate with mitochondrial and nuclear DNA. Our results highlight senescence changes in epithelial cells and how they might contribute to chronic inflammation and age-related diseases.

Increased plasma mitochondrial DNA concentrations are associated with poor outcomes in multiple critical illnesses, including COVID-19. However, current methods of cell-free mitochondrial DNA quantification in plasma are time-consuming and lack reproducibility. Here, we used next-generation sequencing to characterize the size and genome location of circulating mitochondrial DNA in critically ill subjects with COVID-19 to develop a facile and optimal method of quantification by droplet digital PCR. Sequencing revealed a large percentage of small mitochondrial DNA fragments in plasma with wide variability in coverage by genome location. We identified probes for the mitochondrial DNA genes, cytochrome B and NADH dehydrogenase 1, in regions of relatively high coverage that target small sequences potentially missed by other methods. Serial assessments of absolute mitochondrial DNA concentrations were then determined in plasma from 20 critically ill subjects with COVID-19 without a DNA isolation step. Mitochondrial DNA concentrations on the day of enrollment were increased significantly in patients with moderate or severe acute respiratory distress syndrome (ARDS) compared with those with no or mild ARDS. Comparisons of mitochondrial DNA concentrations over time between patients with no/mild ARDS who survived, patients with moderate/severe ARDS who survived, and nonsurvivors showed the highest concentrations in patients with more severe disease. Absolute mitochondrial DNA quantification by droplet digital PCR is time-efficient and reproducible; thus, we provide a valuable tool and rationale for future studies evaluating mitochondrial DNA as a real-time biomarker to guide clinical decision-making in critically ill subjects with COVID-19.

Mutations in VPS13C cause early-onset, autosomal recessive Parkinson's disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the ER and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS-STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation in a model human cell line and place VPS13C in pathways relevant to PD pathogenesis. ispartof: JOURNAL OF CELL BIOLOGY vol:221 issue:7 ispartof: location:United States status: published

SUMMARYMitochondrial DNA (mtDNA) is a potent agonist of the innate immune system; however, the exact immunostimulatory features of mtDNA and the kinetics of mtDNA detection by cytosolic nucleic acid sensors remain poorly defined. Here, we show that mitochondrial genome instability leads to Z-form mtDNA accumulation. Z-DNA Binding Protein 1 (ZBP1) stabilizes Z-form mtDNA and nucleates a cytosolic complex containing cGAS, RIPK1, and RIPK3 to sustain STAT1 phosphorylation and type I interferon (IFN-I) signaling. Increased mitochondrial Z-DNA, ZBP1 expression, and IFN-I responses are observed in cardiomyocytes after exposure to Doxorubicin, a first-line chemotherapeutic agent that induces frequent cardiotoxicity in cancer patients. Strikingly, mice lacking ZBP1 or IFN-I signaling are protected from Doxorubicin-induced cardiotoxicity. Our findings reveal ZBP1 as a cooperative partner for cGAS that sustains IFN-I responses to mitochondrial genome instability and highlight ZBP1 as a potential target in heart failure and other disorders where mtDNA stress contributes to interferon-related pathology.

The actin cytoskeleton plays multiple critical roles in cells, from cell migration to organelle dynamics. The small and transient actin structures regulating organelle dynamics are difficult to detect with fluorescence microscopy, and the limited resolution of fluorescence microscopy makes it difficult to determine whether actin filaments are directly associated with specific membranes. To address these limitations, we developed an approach using fluorescent protein-tagged actin nanobodies targeted to organelle membranes to enable live cell imaging of sub-organellar actin dynamics with unprecedented spatiotemporal resolution.

The recognition that cytosolic mtDNA activates cGAS-STING innate immune signaling has unlocked novel disease mechanisms. Here, an uncharacterized variant predicted to affect TOP1MT function, P193L, was discovered in a family with multiple early-onset autoimmune diseases, including Systemic Lupus Erythematosus (SLE). Although there was no previous genetic association betweenTOP1MTand autoimmune disease, the role of TOP1MT as a regulator of mtDNA led us to investigate whether TOP1MT could mediate the release of mtDNA to the cytosol, where it could then activate the cGAS-STING innate immune pathway known to be activated in SLE and other autoimmune diseases. Through analysis of cells with reduced TOP1MT expression, we show that loss of TOP1MT results in release of mtDNA to the cytosol, which activates the cGAS-STING pathway. We also characterized the P193L variant for its ability to rescue several TOP1MT functions when expressed inTOP1MTknockout cells. We show that the P193L variant is not fully functional, as its re-expression at high levels was unable to rescue mitochondrial respiration deficits, and only showed partial rescue for other functions, including repletion of mtDNA replication following depletion, nucleoid size, steady state mtDNA transcripts levels, and mitochondrial morphology. Additionally, expression of P193L at endogenous levels was unable to rescue mtDNA release-mediated cGAS-STING signaling. Overall, we report a link between TOP1MT and mtDNA release leading to cGAS-STING activation. Moreover, we show that the P193L variant has partial loss of function that may contribute to autoimmune disease susceptibility via cGAS-STING mediated activation of the innate immune system.

Using mitochondria-targeted TALENS and ionizing radiation, consequences of mtDNA double-strand (ds) breaks were investigated by Tigano et al. (2021) who uncovered mtRNA as a retrograde second messenger of this form of mtDNA stress that activates the RIG-I/MAVS innate immune signaling pathway.

Understanding basic mechanisms of aging holds great promise for developing interventions that prevent or delay many age-related declines and diseases simultaneously to increase human healthspan. However, a major confounding factor in aging research is the heterogeneity of the aging process itself. At the organismal level, it is clear that chronological age does not always predict biological age or susceptibility to frailty or pathology. While genetics and environment are major factors driving variable rates of aging, additional complexity arises because different organs, tissues, and cell types are intrinsically heterogeneous and exhibit different aging trajectories normally or in response to the stresses of the aging process (e.g., damage accumulation). Tackling the heterogeneity of aging requires new and specialized tools (e.g., single-cell analyses, mass spectrometry-based approaches, and advanced imaging) to identify novel signatures of aging across scales. Cutting-edge computational approaches are then needed to integrate these disparate datasets and elucidate network interactions between knownaging hallmarks. There is also a need for improved, human cell-based models of aging to ensure that basic research findings are relevant to human aging and healthspan interventions. The San Diego Nathan Shock Center (SD-NSC) provides access to cutting-edge scientific resources to facilitate the study of the heterogeneity of aging in general and to promote the use of novel human cell models of aging. The center also has a robust Research Development Core that funds pilot projects on the heterogeneity of aging and organizes innovative training activities, including workshops and a personalized mentoring program, to help investigators new to the aging field succeed. Finally, the SD-NSC participates in outreach activities to educate the general community about the importance of aging research and promote the need for basic biology of aging research in particular.

This method describes isolation of RNA from cultured cells, generation of cDNA, and relative quantification of transcript levels by qPCR.

This protocol describes the purification of a cytosolic fraction depleted of membrane from cultured cells, and the quantification of mitochondrial DNA (mtDNA) in this fraction by qPCR.

Deleterious somatic mutations in DNA methyltransferase 3 alpha (DNMT3A) and TET mehtylcytosine dioxygenase 2 (TET2) are associated with clonal expansion of hematopoietic cells and higher risk of cardiovascular disease (CVD). Here, we investigated roles of DNMT3A and TET2 in normal human monocyte-derived macrophages (MDM), in MDM isolated from individuals with DNMT3A or TET2 mutations, and in macrophages isolated from human atherosclerotic plaques. We found that loss of function of DNMT3A or TET2 resulted in a type I interferon response due to impaired mitochondrial DNA integrity and activation of cGAS signaling. DNMT3A and TET2 normally maintained mitochondrial DNA integrity by regulating the expression of transcription factor A mitochondria (TFAM) dependent on their interactions with RBPJ and ZNF143 at regulatory regions of the TFAM gene. These findings suggest that targeting the cGAS-type I IFN pathway may have therapeutic value in reducing risk of CVD in patients with DNMT3A or TET2 mutations.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1β (IL-1β) production and mtDNA release in mice.

Mutations in VPS13C cause early onset, autosomal recessive Parkinson’s Disease (PD). We have established that VPS13C encodes a lipid transfer protein localized to contact sites between the endoplasmic reticulum (ER) and late endosomes/lysosomes. In the current study, we demonstrate that depleting VPS13C in HeLa cells causes an accumulation of lysosomes with an altered lipid profile, including an accumulation of di-22:6-BMP, a biomarker of the PD-associated leucine-rich repeat kinase 2 (LRRK2) G2019S mutation. In addition, the DNA-sensing cGAS/STING pathway, which was recently implicated in PD pathogenesis, is activated in these cells. This activation results from a combination of elevated mitochondrial DNA in the cytosol and a defect in the degradation of activated STING, a lysosome-dependent process. These results suggest a link between ER-lysosome lipid transfer and innate immune activation and place VPS13C in pathways relevant to PD pathogenesis.

Coronavirus disease 2019 (COVID-19) includes the cardiovascular complications in addition to respiratory disease. SARS-CoV-2 infection impairs endothelial function and induces vascular inflammation, leading to endotheliitis. SARS-CoV-2 infection relies on the binding of Spike glycoprotein (S protein) to angiotensin converting enzyme 2 (ACE2) in the host cells. We show here that S protein alone can damage vascular endothelial cells (ECs) in vitro and in vivo, manifested by impaired mitochondrial function, decreased ACE2 expression and eNOS activity, and increased glycolysis. The underlying mechanism involves S protein downregulation of AMPK and upregulation of MDM2, causing ACE2 destabilization. Thus, the S protein-exerted vascular endothelial damage via ACE2 downregulation overrides the decreased virus infectivity.

The mammalian genome comprises nuclear DNA (nDNA) derived from both parents and mitochondrial DNA (mtDNA) that is maternally inherited and encodes essential proteins required for oxidative phosphorylation. Thousands of copies of the circular mtDNA are present in most cell types that are packaged by TFAM into higher-order structures called nucleoids1. Mitochondria are also platforms for antiviral signalling2 and, due to their bacterial origin, mtDNA and other mitochondrial components trigger innate immune responses and inflammatory pathology2,3. We showed previously that cytoplasmic release of mtDNA activates the cGAS–STING–TBK1 pathway resulting in interferon-stimulated gene (ISG) expression that promotes antiviral immunity4. Here, we find that persistent mtDNA stress is not associated with basally activated NF-κB signalling or interferon gene expression typical of an acute antiviral response. Instead, a specific subset of ISGs that includes Parp9 remains activated by the unphosphorylated form of ISGF3 that enhances nDNA damage and repair responses. In cultured primary fibroblasts and cancer cells, the chemotherapeutic drug doxorubicin causes mtDNA damage and release, which leads to cGAS–STING–dependent ISG activation. In addition, mtDNA stress in TFAM-deficient mouse melanoma cells produces tumours that are more resistant to doxorubicin in vivo. Finally, Tfam+/− mice exposed to ionizing radiation exhibit enhanced nDNA repair responses in spleen. Therefore, we propose that damage to and subsequent release of mtDNA elicits a protective signalling response that enhances nDNA repair in cells and tissues, suggesting that mtDNA is a genotoxic stress sentinel. Persistent mitochondrial DNA stress is shown to upregulate nuclear DNA damage and repair responses via activation of the cGAS–STING pathway and a subset of interferon-stimulated genes.

Mitochondrial protein interactions and complexes facilitate mitochondrial function. These complexes range from simple dimers to the respirasome supercomplex consisting of oxidative phosphorylation complexes I, III, and IV. To improve understanding of mitochondrial function, we used chemical cross-linking mass spectrometry to identify 2,427 cross-linked peptide pairs from 327 mitochondrial proteins in whole, respiring murine mitochondria. In situ interactions were observed in proteins throughout the electron transport chain membrane complexes, ATP synthase, and the mitochondrial contact site and cristae organizing system (MICOS) complex. Cross-linked sites showed excellent agreement with empirical protein structures and delivered complementary constraints for in silico protein docking. These data established direct physical evidence of the assembly of the complex I-III respirasome and enabled prediction of in situ interfacial regions of the complexes. Finally, we established a database and tools to harness the cross-linked interactions we observed as molecular probes, allowing quantification of conformation-dependent protein interfaces and dynamic protein complex assembly.

Newman and Shadel preview new work from the Youle laboratory published in Nature indicating that mitophagy mitigates STING-induced inflammation., What causes inflammation in age-related neurodegenerative diseases remains a mystery. Sliter et al. (2018. Nature. https://doi.org/10.1038/s41586-018-0448-9) show that, when damaged mitochondria cannot be removed by mitophagy, stress from exercise or mitochondrial DNA mutations activates the proinflammatory cGAS–STING pathway that may contribute to Parkinson’s disease.

Chronic obstructive pulmonary disease (COPD) encompasses several clinical syndromes, most notably emphysema and chronic bronchitis. Most of the current treatments fail to attenuate severity and progression of the disease, thereby requiring better mechanistic understandings of pathogenesis to develop disease-modifying therapeutics. A number of theories on COPD pathogenesis have been promulgated wherein an increase in protease burden from chronic inflammation, exaggerated production of reactive oxygen species and the resulting oxidant injury, or superfluous cell death responses caused by enhanced cellular injury/damage were proposed as the culprit. These hypotheses are not mutually exclusive and together likely represent the multifaceted biological processes involved in COPD pathogenesis. Recent studies demonstrate that mitochondria are involved in innate immune signaling that plays important roles in cigarette smoke-induced inflammasome activation, pulmonary inflammation and tissue remodeling responses. These responses are reviewed herein and synthesized into a view of COPD pathogenesis whereby mitochondria play a central role.

// Amanda R. Oran 1 , Clare M. Adams 1 , Xiao-yong Zhang 1 , Victoria J. Gennaro 1 , Harla K. Pfeiffer 1 , Hestia S. Mellert 2 , Hans E. Seidel 3 , Kirsten Mascioli 1 , Jordan Kaplan 1 , Mahmoud R. Gaballa 1 , Chen Shen 4,5 , Isidore Rigoutsos 1 , Michael P. King 6 , Justin L. Cotney 7 , Jamie J. Arnold 8 , Suresh D. Sharma 8 , Ubaldo E. Martinez-Outschoorn 1 , Christopher R. Vakoc 4 , Lewis A. Chodosh 3 , James E. Thompson 9 , James E. Bradner 10 , Craig E. Cameron 8 , Gerald S. Shadel 11,12 , Christine M. Eischen 1 and Steven B. McMahon 1 1 Department of Cancer Biology, Thomas Jefferson University, Philadelphia, PA, USA 2 Biomedical Graduate Studies, University of Pennsylvania, Philadelphia, PA, USA 3 Department of Cancer Biology and Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA 4 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA 5 Molecular and Cellular Biology Program, Stony Brook University, Stony Brook, NY, USA 6 Department of Biochemistry, Thomas Jefferson University, Philadelphia, PA, USA 7 Genetics and Genome Sciences, University of Connecticut Health, Farmington, CT, USA 8 Department of Biochemistry & Molecular Biology, The Pennsylvania State University, University Park, PA, USA 9 Leukemia Service, Department of Medicine, Roswell Park Cancer Institute, Buffalo, NY, USA 10 Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA,USA 11 Department of Pathology, Yale School of Medicine, New Haven, CT, USA 12 Department of Genetics, Yale School of Medicine, New Haven, CT, USA Correspondence to: Steven B. McMahon, email: // Keywords : MYC, mitochondria, mitochondrial gene expression, tigecycline, synthetic lethality Received : June 08, 2016 Accepted : August 25, 2016 Published : August 31, 2016 Abstract Despite ubiquitous activation in human cancer, essential downstream effector pathways of the MYC transcription factor have been difficult to define and target. Using a structure/function-based approach, we identified the mitochondrial RNA polymerase (POLRMT) locus as a critical downstream target of MYC. The multifunctional POLRMT enzyme controls mitochondrial gene expression, a process required both for mitochondrial function and mitochondrial biogenesis. We further demonstrate that inhibition of this newly defined MYC effector pathway causes robust and selective tumor cell apoptosis, via an acute, checkpoint-like mechanism linked to aberrant electron transport chain complex assembly and mitochondrial reactive oxygen species (ROS) production. Fortuitously, MYC-dependent tumor cell death can be induced by inhibiting the mitochondrial gene expression pathway using a variety of strategies, including treatment with FDA-approved antibiotics. In vivo studies using a mouse model of Burkitt’s Lymphoma provide pre-clinical evidence that these antibiotics can successfully block progression of MYC-dependent tumors.

To translate the 13 mtDNA-encoded mRNAs involved in oxidative phosphorylation (OXPHOS), mammalian mitochondria contain a dedicated set of ribosomes comprising rRNAs encoded by the mitochondrial genome and mitochondrial ribosomal proteins (MRPs) that are encoded by nuclear genes and imported into the matrix. In addition to their role in the ribosome, several MRPs have auxiliary functions or have been implicated in other cellular processes like cell cycle regulation and apoptosis. For example, we have shown that human MRPL12 binds and activates mitochondrial RNA polymerase (POLRMT), and hence has distinct functions in the ribosome and mtDNA transcription. Here we provide concrete evidence that there are two mature forms of mammalian MRPL12 that are generated by a two-step cleavage during import, involving efficient cleavage by mitochondrial processing protease and a second inefficient or regulated cleavage by mitochondrial intermediate protease. We also show that knock-down of MRPL12 by RNAi results in instability of POLRMT, but not other primary mitochondrial transcription components, and a corresponding decrease in mitochondrial transcription rates. Knock-down of MRPL10, the binding partner of MRPL12 in the ribosome, results in selective degradation of the mature long form of MRPL12, but has no effect on POLRMT. We propose that the two forms of MRPL12 are involved in homeostatic regulation of mitochondrial transcription and ribosome biogenesis that likely contribute to cell cycle, growth regulation, and longevity pathways to which MRPL12 has been linked.

[EN] Hutchinson-Gilford progeria syndrome (HGPS) is an ultra-rare disorder with devastating sequelae resulting in early death, presently thought to stem primarily from cardiovascular events. We analyse novel longitudinal cardiovascular data from a mouse model of HGPS (Lmna(G609G/G609G)) using allometric scaling, biomechanical phenotyping, and advanced computational modelling and show that late-stage diastolic dysfunction, with preserved systolic function, emerges with an increase in the pulse wave velocity and an associated loss of aortic function, independent of sex. Specifically, there is a dramatic late-stage loss of smooth muscle function and cells and an excessive accumulation of proteoglycans along the aorta, which result in a loss of biomechanical function (contractility and elastic energy storage) and a marked structural stiffening despite a distinctly low intrinsic material stiffness that is consistent with the lack of functional lamin A. Importantly, the vascular function appears to arise normally from the low-stress environment of development, only to succumb progressively to pressure-related effects of the lamin A mutation and become extreme in the peri-morbid period. Because the dramatic life-threatening aortic phenotype manifests during the last third of life there may be a therapeutic window in maturity that could alleviate concerns with therapies administered during early periods of arterial development., This work was supported, in part, by grants from the US National Institutes of Health: R01 HL105297 (J.D.H.) and P01 HL134605 (Dan Rifkin) and R01 AG047632 and R33 ES025636 (G.S.S.)

Acquired resistance to HER2-targeted therapies occurs frequently in HER2+ breast tumors and new strategies for overcoming resistance are needed. Here, we report that resistance to trastuzumab is reversible, as resistant cells regained sensitivity to the drug after being cultured in drug-free media. RNA-sequencing analysis showed that cells resistant to trastuzumab or trastuzumab + pertuzumab in combination increased expression of oxidative phosphorylation pathway genes. Despite minimal changes in mitochondrial respiration, these cells exhibited increased expression of ATP synthase genes and selective dependency on ATP synthase function. Resistant cells were sensitive to inhibition of ATP synthase by oligomycin A, and knockdown of ATP5J or ATP5B, components of ATP synthase complex, rendered resistant cells responsive to a low dose of trastuzumab. Furthermore, combining ATP synthase inhibitor oligomycin A with trastuzumab led to regression of trastuzumab-resistant tumors in vivo. In conclusion, we identify a novel vulnerability of cells with acquired resistance to HER2-targeted antibody therapies and reveal a new therapeutic strategy to overcome resistance. Significance: These findings implicate ATP synthase as a novel potential target for tumors resistant to HER2-targeted therapies.

Mitochondria are integral to cellular energy metabolism and ATP production, and are involved in regulating many cellular processes. Mitochondria produce reactive oxygen species (ROS) that can damage cellular components but that also participate in signal transduction. The kinase ATM, which is mutated in the neurodegenerative, autosomal recessive disease Ataxia-Telangiectasia (A-T), is a key player in the nuclear DNA-damage response. However, ATM also performs a redox-sensing function mediated by its ROS-dependent formation of disulfide-linked dimers. Here, we found that mitochondria-derived hydrogen peroxide promoted ATM redox dimerization. In HeLa cells, ATM dimers were localized to the nucleus and inhibited by the redox regulatory protein thioredoxin 1 (TRX1), suggesting the existence of a ROS-mediated, stress-signaling relay from mitochondria to the nucleus. ATM dimer formation did not affect its association with chromatin in the absence or presence of nuclear DNA damage, consistent with separation of its redox and DNA-damage signaling functions. Comparative analysis of U2OS cells expressing either wild-type ATM or the redox-sensing-deficient C2991L mutant revealed that one function of ATM redox-sensing is to promote glucose flux through the pentose phosphate pathway (PPP) by increasing the expression and activity of glucose-6-phosphate dehydrogenase (G6PD), thereby increasing cellular antioxidant capacity. The PPP produces the coenzyme NADPH needed for a robust antioxidant response, including the regeneration of TRX1, indicating the existence of a regulatory feedback loop involving ATM and TRX1. We propose that loss of the mitochondrial ROS-sensing function of ATM may cause cellular ROS accumulation and oxidative stress in A-T.

Cyclic GMP-AMP (cGAMP) synthase (cGAS) stimulator of interferon genes (STING) senses pathogen-derived or abnormal self-DNA in the cytosol and triggers an innate immune defense against microbial infection and cancer. STING agonists induce both innate and adaptive immune responses and are a new class of cancer immunotherapy agents tested in multiple clinical trials. However, STING is commonly silenced in cancer cells via unclear mechanisms, limiting the application of these agonists. Here, we report that the expression of STING is epigenetically suppressed by the histone H3K4 lysine demethylases KDM5B and KDM5C and is activated by the opposing H3K4 methyltransferases. The induction of STING expression by KDM5 blockade triggered a robust interferon response in a cytosolic DNA-dependent manner in breast cancer cells. This response resulted in resistance to infection by DNA and RNA viruses. In human tumors, KDM5B expression is inversely associated with STING expression in multiple cancer types, with the level of intratumoral CD8+ T cells, and with patient survival in cancers with a high level of cytosolic DNA, such as human papilloma virus (HPV)-positive head and neck cancer. These results demonstrate a novel epigenetic regulatory pathway of immune response and suggest that KDM5 demethylases are potential targets for antipathogen treatment and anticancer immunotherapy., Author summary Pathogens often find ways to turn down cell-intrinsic antipathogen immune responses by the host. Similarly, cancer cells use various mechanisms to evade attack by immune cells. One of the common mechanisms is suppression of the stimulator of interferon genes (STING)-dependent innate immune response. Using potent and specific small-molecule inhibitors and genetic-depletion approaches, we found that the silenced STING pathway can be reactivated in breast cancer cells by suppressing KDM5 demethylases. Activation of the STING pathway led to a robust interferon response, which blocked viral infection, and was associated with increased tumor-infiltrated lymphocytes and better patient survival in multiple cancer types. This discovery has major clinical implications for treating both pathogen infection and cancer because KDM5 inhibition provides a fast, robust, and reversible control of innate immune response. Since the discovery of histone demethylase activity of KDM5 proteins a decade ago, significant efforts have been dedicated to developing KDM5 inhibitors for clinical applications. In fact, a KDM5 inhibitor recently entered phase I clinical trial for treatment of hepatitis B infection. Here, we provide mechanistic insights on how KDM5 inhibitors block viral infection. Moreover, our results suggest that KDM5 inhibitors can also be combined with other cancer immunotherapies.

Dietary lipid overload and calorie excess during obesity is a low grade chronic inflammatory state with diminished ability to appropriately metabolize glucose or lipids. Macrophages are critical in maintaining adipose tissue homeostasis, in part by regulating lipid metabolism, energy homeostasis, and tissue remodeling. During high fat diet-induced obesity, macrophages are activated by lipid derived "danger signals" such as ceramides and palmitate and promote the adipose tissue inflammation in an Nlrp3 inflammasome-dependent manner. Given that the metabolic fate of fatty acids in macrophages is not entirely elucidated, we have hypothesized that de novo synthesis of ceramide, through the rate-limiting enzyme serine palmitoyltransferase long chain (Sptlc)-2, is required for saturated fatty acid-driven Nlrp3 inflammasome activation in macrophages. Here we report that mitochondrial targeted overexpression of catalase, which is established to mitigate oxidative stress, controls ceramide-induced Nlrp3 inflammasome activation but does not affect the ATP-mediated caspase-1 cleavage. Surprisingly, myeloid cell-specific deletion of Sptlc2 is not required for palmitate-driven Nlrp3 inflammasome activation. Furthermore, the ablation of Sptlc2 in macrophages did not impact macrophage polarization or obesity-induced adipose tissue leukocytosis. Consistent with these data, investigation of insulin resistance using hyperinsulinemic-euglycemic clamps revealed no significant differences in obese mice lacking ceramide de novo synthesis machinery in macrophages. These data suggest that alternate metabolic pathways control fatty acid-derived ceramide synthesis in macrophage and the Nlrp3 inflammasome activation in obesity.

Cigarette smoke (CS) and viruses promote the inflammation and remodeling associated with chronic obstructive pulmonary disease (COPD). The MAVS/RIG-I–like helicase (MAVS/RLH) pathway and inflammasome-dependent innate immune pathways are important mediators of these responses. At baseline, the MAVS/RLH pathway is suppressed, and this inhibition must be reversed to engender tissue effects; however, the mechanisms that mediate activation and repression of the pathway have not been defined. In addition, the regulation and contribution of MAVS/RLH signaling in CS-induced inflammation and remodeling responses and in the development of human COPD remain unaddressed. Here, we demonstrate that expression of NLRX1, which inhibits the MAVS/RLH pathway and regulates other innate immune responses, was markedly decreased in 3 independent cohorts of COPD patients. NLRX1 suppression correlated directly with disease severity and inversely with pulmonary function, quality of life, and prognosis. In murine models, CS inhibited NLRX1, and CS-induced inflammation, alveolar destruction, protease induction, structural cell apoptosis, and inflammasome activation were augmented in NLRX1-deficient animals. Conversely, MAVS deficiency abrogated this CS-induced inflammation and remodeling. Restoration of NLRX1 in CS-exposed animals ameliorated alveolar destruction. These data support a model in which CS-dependent NLRX1 inhibition facilitates MAVS/RHL activation and subsequent inflammation, remodeling, protease, cell death, and inflammasome responses.

Mitochondrial DNA (mtDNA) is normally present at thousands of copies per cell and is packaged into several hundred higher-order structures termed nucleoids. The abundant mtDNA-binding protein TFAM (transcription factor A, mitochondrial) regulates nucleoid architecture, abundance and segregation. Complete mtDNA depletion profoundly impairs oxidative phosphorylation, triggering calcium-dependent stress signalling and adaptive metabolic responses. However, the cellular responses to mtDNA instability, a physiologically relevant stress observed in many human diseases and ageing, remain poorly defined. Here we show that moderate mtDNA stress elicited by TFAM deficiency engages cytosolic antiviral signalling to enhance the expression of a subset of interferon-stimulated genes. Mechanistically, we find that aberrant mtDNA packaging promotes escape of mtDNA into the cytosol, where it engages the DNA sensor cGAS (also known as MB21D1) and promotes STING (also known as TMEM173)-IRF3-dependent signalling to elevate interferon-stimulated gene expression, potentiate type I interferon responses and confer broad viral resistance. Furthermore, we demonstrate that herpesviruses induce mtDNA stress, which enhances antiviral signalling and type I interferon responses during infection. Our results further demonstrate that mitochondria are central participants in innate immunity, identify mtDNA stress as a cell-intrinsic trigger of antiviral signalling and suggest that cellular monitoring of mtDNA homeostasis cooperates with canonical virus sensing mechanisms to fully engage antiviral innate immunity.

To elucidate gene expression pathways underlying age-associated impairment in influenza vaccine response, we screened young (age 21-30) and older (age ≥65) adults receiving influenza vaccine in two consecutive seasons and identified those with strong or absent response to vaccine, including a subset of older adults meeting criteria for frailty. PBMCs obtained prior to vaccination (Day 0) and at day 2 or 4, day 7 and day 28 post-vaccine were subjected to gene expression microarray analysis. We defined a response signature and also detected induction of a type I interferon response at day 2 and a plasma cell signature at day 7 post-vaccine in young responders. The response signature was dysregulated in older adults, with the plasma cell signature induced at day 2, and was never induced in frail subjects (who were all non-responders). We also identified a mitochondrial signature in young vaccine responders containing genes mediating mitochondrial biogenesis and oxidative phosphorylation that was consistent in two different vaccine seasons and verified by analyses of mitochondrial content and protein expression. These results represent the first genome-wide transcriptional profiling analysis of age-associated dynamics following influenza vaccination, and implicate changes in mitochondrial biogenesis and function as a critical factor in human vaccine responsiveness.

Mitochondrial DNA (mtDNA) - which is well known for its role in oxidative phosphorylation and maternally inherited mitochondrial diseases - is increasingly recognized as an agonist of the innate immune system that influences antimicrobial responses and inflammatory pathology. On entering the cytoplasm, extracellular space or circulation, mtDNA can engage multiple pattern-recognition receptors in cell-type- and context-dependent manners to trigger pro-inflammatory and type I interferon responses. Here, we review the expanding research field of mtDNA in innate immune responses to highlight new mechanistic insights and discuss the physiological and pathological relevance of this exciting area of mitochondrial biology.

SummaryThe mechanism by which cells undergo death determines whether dying cells trigger inflammatory responses or remain immunologically silent. Mitochondria play a central role in the induction of cell death, as well as in immune signaling pathways. Here, we identify a mechanism by which mitochondria and downstream proapoptotic caspases regulate the activation of antiviral immunity. In the absence of active caspases, mitochondrial outer membrane permeabilization by Bax and Bak results in the expression of type I interferons (IFNs). This induction is mediated by mitochondrial DNA-dependent activation of the cGAS/STING pathway and results in the establishment of a potent state of viral resistance. Our results show that mitochondria have the capacity to simultaneously expose a cell-intrinsic inducer of the IFN response and to inactivate this response in a caspase-dependent manner. This mechanism provides a dual control, which determines whether mitochondria initiate an immunologically silent or a proinflammatory type of cell death.

Sepsis is a systemic inflammatory response to infection and a major cause of death worldwide. Because specific therapies to treat sepsis are limited, and underlying pathogenesis is unclear, current medical care remains purely supportive. Therefore targeted therapies to treat sepsis need to be developed. Although an important mediator of sepsis is thought to be mitochondrial dysfunction, the underlying molecular mechanism is unclear. Modulation of mitochondrial processes may be an effective therapeutic strategy in sepsis. Here, we investigated the role of the kinase MKK3 in regulation of mitochondrial function in sepsis. Using clinically relevant animal models, we examined mitochondrial function in primary mouse lung endothelial cells exposed to LPS. MKK3 deficiency reduces lethality of sepsis in mice and by lowering levels of lung and mitochondrial injury as well as reactive oxygen species. Furthermore, MKK3 deficiency appeared to simultaneously increase mitochondrial biogenesis and mitophagy through the actions of Sirt1, Pink1, and Parkin. This led to a more robust mitochondrial network, which we propose provides protection against sepsis. We also detected higher MKK3 activation in isolated peripheral blood mononuclear cells from septic patients compared with nonseptic controls. Our findings demonstrate a critical role for mitochondria in the pathogenesis of sepsis that involves a previously unrecognized function of MKK3 in mitochondrial quality control. This mitochondrial pathway may help reveal new diagnostic markers and therapeutic targets against sepsis.

Cellular homeostasis is coordinated through communication between mitochondria and the nucleus, organelles that each possess their own genomes. Whereas the mitochondrial genome is regulated by factors encoded in the nucleus, the nuclear genome is currently not known to be actively controlled by factors encoded in the mitochondrial DNA. Here, we show that MOTS-c, a peptide encoded in the mitochondrial genome, translocates to the nucleus and regulates nuclear gene expression following metabolic stress in a 5′-adenosine mono-phosphate-activated protein kinase (AMPK)-dependent manner. In the nucleus, MOTS-c regulated a broad range of genes in response to glucose restriction, including those with antioxidant response elements (ARE), and interacted with ARE-regulating stress-responsive transcription factors, such as nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2). Our findings indicate that the mitochondrial and nuclear genomes co-evolved to independently encode for factors to cross-regulate each other, suggesting that mitonuclear communication is genetically integrated.

Summary Immunotherapy has revolutionized cancer treatment, yet most patients do not respond. Here, we investigated mechanisms of response by profiling the proteome of clinical samples from advanced stage melanoma patients undergoing either tumor infiltrating lymphocyte (TIL)-based or anti- programmed death 1 (PD1) immunotherapy. Using high-resolution mass spectrometry, we quantified over 10,300 proteins in total and ∼4,500 proteins across most samples in each dataset. Statistical analyses revealed higher oxidative phosphorylation and lipid metabolism in responders than in non-responders in both treatments. To elucidate the effects of the metabolic state on the immune response, we examined melanoma cells upon metabolic perturbations or CRISPR-Cas9 knockouts. These experiments indicated lipid metabolism as a regulatory mechanism that increases melanoma immunogenicity by elevating antigen presentation, thereby increasing sensitivity to T cell mediated killing both in vitro and in vivo. Altogether, our proteomic analyses revealed association between the melanoma metabolic state and the response to immunotherapy, which can be the basis for future improvement of therapeutic response.

SummaryReactive oxygen species (ROS) play complex roles in aging, having both damaging effects and signaling functions. Transiently elevating mitochondrial stress, including mitochondrial ROS (mtROS), elicits beneficial responses that extend lifespan. However, these adaptive, longevity-signaling pathways remain poorly understood. We show here that Tel1p and Rad53p, homologs of the mammalian DNA damage response kinases ATM and Chk2, mediate a hormetic mtROS longevity signal that extends yeast chronological lifespan. This pathway senses mtROS in a manner distinct from the nuclear DNA damage response and ultimately imparts longevity by inactivating the histone demethylase Rph1p specifically at subtelomeric heterochromatin, enhancing binding of the silencing protein Sir3p, and repressing subtelomeric transcription. These results demonstrate the existence of conserved mitochondria-to-nucleus stress-signaling pathways that regulate aging through epigenetic modulation of nuclear gene expression.

The human genome comprises large chromosomes in the nucleus and mitochondrial DNA (mtDNA) housed in the dynamic mitochondrial network. Human cells contain up to thousands of copies of the double-stranded, circular mtDNA molecule that encodes essential subunits of the oxidative phosphorylation complexes and the rRNAs and tRNAs needed to translate these in the organelle matrix. Transcription of human mtDNA is directed by a single-subunit RNA polymerase, POLRMT, which requires two primary transcription factors, TFB2M (transcription factor B2, mitochondrial) and TFAM (transcription factor A, mitochondrial), to achieve basal regulation of the system. Here, we review recent advances in understanding the structure and function of the primary human transcription machinery and the other factors that facilitate steps in transcription beyond initiation and provide more intricate control over the system.