Mitonuclear Protein Imbalance Research Articles

Diterpenoid honatisine overcomes temozolomide resistance in glioblastoma by inducing mitonuclear protein imbalance through disruption of TFAM-mediated mtDNA transcription

BackgroundGlioblastoma (GBM) represents as the most formidable intracranial malignancy. The systematic exploration of natural compounds for their potential applications in GBM therapy has emerged as a pivotal and fruitful avenue of research. PurposeIn the present study, a panel of 96 diterpenoids was systematically evaluated as a repository of potential antitumour agents. The primary objective was to discern their potency in overcoming resistance to temozolomide (TMZ). Through an extensive screening process, honatisine, a heptacyclic diterpenoid alkaloid, emerged as the most robust candidate. Notably, honatisine exhibited remarkable efficacy in patient-derived primary and recurrent GBM strains. Subsequently, we subjected this compound to comprehensive scrutiny, encompassing GBM cultured spheres, GBM organoids (GBOs), TMZ-resistant GBM cell lines, and orthotopic xenograft mouse models of GBM cells. ResultsOur investigative efforts delved into the mechanistic underpinnings of honatisine's impact. It was discerned that honatisine prompted mitonuclear protein imbalance and elicited the mitochondrial unfolded protein response (UPRmt). This effect was mediated through the selective depletion of mitochondrial DNA (mtDNA)-encoded subunits, with a particular emphasis on the diminution of mitochondrial transcription factor A (TFAM). The ultimate outcome was the instigation of deleterious mitochondrial dysfunction, culminating in apoptosis. Molecular docking and surface plasmon resonance (SPR) experiments validated honatisine's binding affinity to TFAM within its HMG-box B domain. This binding may promote phosphorylation of TFAM and obstruct the interaction of TFAM bound to heavy strand promoter 1 (HSP1), thereby enhancing Lon-mediated TFAM degradation. Finally, in vivo experiments confirmed honatisine's antiglioma properties. Our comprehensive toxicological assessments underscored its mild toxicity profile, emphasizing the necessity for a thorough evaluation of honatisine as a novel antiglioma agent. ConclusionIn summary, our data provide new insights into the therapeutic mechanisms underlying honatisine's selective inducetion of apoptosis and its ability to overcome chemotherapy resistance in GBM. These actions are mediated through the disruption of mitochondrial proteostasis and function, achieved by the inhibition of TFAM-mediated mtDNA transcription. This study highlights honatisine's potential as a promising agent for glioblastoma therapy, underscoring the need for further exploration and investigation.

Reduced Mitochondrial Protein Translation Promotes Cardiomyocyte Proliferation and Heart Regeneration.

The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

Dietary cobalt oxide nanoparticles alleviate aging through activation of mitochondrial UPR in Caenorhabditis elegans.

Mitochondrial unfolded protein response (UPRmt), which is a mitochondrial proteostasis pathway, orchestrates an adaptive reprogramming for metabolism homeostasis and organismal longevity. Similar to other defense systems, compromised UPRmt is a feature of several age-related diseases. Here we report that dimercapto succinic acid (DMSA)-modified cobalt oxide nanoparticles (Co3O4 NPs), which have received wide-spread attention in biomedical fields, is a promising UPRmt activator and, more importantly, provides a gate for extending healthy lifespan. Methods: UPRmt activation by Co3O4 NPs was tested in transgenetic Caenorhabditis elegans (C. elegans) specifically expressing UPRmt reporter Phsp-6::GFP, and the underlying mechanism was further validated by mitochondrial morphology, mtDNA/nDNA, metabolism-related genes' expression, mitonuclear protein imbalance, oyxgen assumption and ATP level in C. elegans. Then therapeutic response aganist senescence was monitored by lifespan analysis, lipofusin contents, MDA contents, Fe accumulation, pharyngeal locomotion performance as well as athletic ability (head thrashes and body bends) at different developmental stages of C. elegans. RNAi towards ubl-5 or atfs-1 in UPRmt pathway was applied to clarify the role of UPRmt in Co3O4 NPs -mediated anti-aging effects. Finally, the effect of Co3O4 NPs on mitochondrial homeostasis and D-galactose-induced cell viability decline in mammalian cells were studied. Results: Co3O4 NPs was revealed as a bona fide activator of the UPRmt signaling pathway, through fine-tuning mitochondrial dynamics and inducing a stoichiometric imbalance between OXPHOS subunits encoded by mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) at early life stage of C. elegans. Phenotypically, Co3O4 NPs treatment protect C. elegans from external stresses. More importantly, dietary low level of Co3O4 NPs effectively extend lifespan and alleviate aging-related physiological and functional decline of worms, demonstrating its potential roles in delaying aging. While the protective effect exerted by Co3O4 NPs was compromised in line with atfs-1 or ubl-5 RNAi treatment. Further studies verified the conservation of Co3O4 NPs in activating UPRmt and exerting protective effects in mammalian cells. Conclusions: The results reveal beneficial effects of Co3O4 NPs on mitochondrial metabolic control, thus presenting their potential efficacy in anti-aging care.

Mitochondrial proteotoxic stresses activate abscisic acid signaling in plants

The mitochondrial proteome is composed of both mitochondrial DNA (mtDNA)-encoded and nuclear DNA (nuDNA)-encoded proteins. The mitonuclear protein imbalance leads to the occurrence of mitochondrial proteotoxic stresses. As a response to the mitochondrial proteotoxic stress, mitochondrial unfolded protein response (UPRmt) is induced in cells to restore the mitochondrial functions. Although UPRmt has been extensively studied in animals, much less is known about the mechanisms of UPRmt signaling in plants. We report here that a DNA intercalating agent, ethidium bromide (EtBr) triggers UPRmt in Arabidopsis thaliana, with characteristic features similar to those caused by the mitochondrial translation inhibitor doxycycline (Dox). EtBr/Dox treatments activate abscisic acid (ABA) signaling. Then ABA promotes the expression of UPRmt genes, like alternative oxidase 1a (AOX1a). A key regulator of ABA signaling, ABA insensitive 5 (ABI5) directly binds to the promoter of AOX1a, which enhances AOX1a gene transcription. Our study reveals a link between ABA and UPRmt in plants.

Heme oxygenase-1 protects against endotoxin-induced acute lung injury depends on NAD+-mediated mitonuclear communication through PGC1α/PPARγ signaling pathway.

Endotoxin-induced acute lung injury (ALI) is a challenging life-threatening disease for which no specific therapy exists. Mitochondrial dysfunction is corroborated as hallmarks in sepsis which commonly disrupt mitochondria-centered cellular communication networks, especially mitonuclear crosstalk, where the ubiquitous cofactor nicotinamide adenine dinucleotide (NAD+) is essential for mitonuclear communication. Heme oxygenase-1 (HO-1) is critical for maintaining mitochondrial dynamic equilibrium and regulating endoplasmic reticulum (ER) and Golgi stress to alleviating acute lung injury. However, it is unclear whether HO-1 regulates NAD+-mediated mitonuclear communication to exert the endogenous protection during endotoxin-induced ALI. In this study, we observed HO-1 attenuated endotoxin-induced ALI by regulated NAD+ levels and NAD+ affected the mitonuclear communication, including mitonuclear protein imbalance and UPRmt to alleviate lung damage. We also found the protective effect of HO-1 depended on NAD+ and NAD+-mediated mitonuclear communication. Furtherly, the inhibition of the PGC1α/PPARγ signaling exacerbates the septic lung injury by reducing NAD+ levels and repressing the mitonuclear protein imbalance and UPRmt. Altogether, our study certified that HO-1 ameliorated endotoxin-induced acute lung injury by regulating NAD+ and NAD+-mediated mitonuclear communications through PGC1α/PPARγ pathway. The present study provided complementary evidence for the cytoprotective effect of HO-1 as a potential target for preventing and attenuating of endotoxin-induced ALI.

Nicotinamide mononucleotide ameliorates acute lung injury by inducing mitonuclear protein imbalance and activating the UPRmt.

Mitochondria need to interact with the nucleus under homeostasis and stress to maintain cellular demands and nuclear transcriptional programs. Disrupted mitonuclear interaction is involved in many disease processes. However, the role of mitonuclear signaling regulators in endotoxin-induced acute lung injury (ALI) remains unknown. Nicotinamide adenine dinucleotide (NAD+) is closely related to mitonuclear interaction with its central role in mitochondrial metabolism. In the current study, C57BL/6J mice were administrated with lipopolysaccharide 15 mg/kg to induce endotoxin-induced ALI and investigated whether the NAD+ precursor nicotinamide mononucleotide (NMN) could preserve mitonuclear interaction and alleviate ALI. After pretreatment with NMN for 7 days, NAD+ levels in the mitochondrial, nucleus, and total intracellular were significantly increased in endotoxemia mice. Moreover, supplementation of NMN alleviated lung pathologic injury, reduced ROS levels, increased MnSOD activities, mitigated mitochondrial dysfunction, ameliorated the defects in the nucleus morphology, and these cytoprotective effects were accompanied by preserving mitonuclear interaction (including mitonuclear protein imbalance and the mitochondrial unfolded protein response, UPRmt). Furthermore, NAD+-mediated mitonuclear protein imbalance and UPRmt are probably regulated by deacetylase Sirtuin1 (SIRT1). Taken together, our results indicated that NMN pretreatment ameliorated ALI by inducing mitonuclear protein imbalance and activating the UPRmt in an SIRT1-dependent manner.

Targeting Mitochondrial Translation Overcomes Venetoclax Resistance in Acute Myeloid Leukemia (AML) through Activation of the Integrated Stress Response

Venetoclax is a highly specific BCL-2 inhibitor with promising activity against AML including leukemic stem cells (LSCs). However, venetoclax monotherapy in AML patients had limited clinical efficacy due to intrinsic or acquired drug resistance which has been attributed to the overexpression of other anti-apoptotic proteins including MCL-1 and BCL-xL. These findings highlight the importance of combination therapy to overcome resistance but the optimal strategy to achieving this goal is unclear.To address this issue, we generated a panel of highly resistant clones derived from the intrinsically sensitive MOLM-13 and MV-4-11 AML cell lines. Surprisingly, treatment with MCL-1 or BCL-xL inhibitors failed to overcome resistance despite higher expression of the anti-apoptotic proteins in resistant cells. To identify novel targets, we performed a genome-wide CRISPR knockout screen to find genes that upon inactivation, restore sensitivity to venetoclax. We transduced one of the resistant clones engineered to stably express Cas9 with a pooled lentiviral single-guide RNA (sgRNA) library that targets 17,661 protein-coding genes with 91,320 unique sgRNA sequences. Transduced cells were divided into 2 populations with one treated with venetoclax and the other untreated as control. The transduced cells were cultured for 29 days to negatively select sgRNAs that re-sensitized resistant cells to venetoclax. Cell samples were collected at regular intervals and subjected to next-generation sequencing of the sgRNA target region to quantify the abundance of each construct.We identified genes that were negatively selected in the presence of venetoclax using the MAGeCK algorithm. The top-ranked genes were highly enriched for gene ontology terms related to mitochondrial translation. We confirmed that RNAi-mediated knockdown of the top-ranked ribosomal subunit gene, DAP3, overcame venetoclax resistance. Pharmacologic inhibition of mitochondrial translation with a panel of protein synthesis inhibitor antibiotics similarly restored sensitivity in AML cell lines with intrinsic or acquired resistance. Tedizolid, a FDA-approved second generation oxazolidinone antibiotic, was the most effective in mediating this effect at clinically relevant concentrations. Furthermore, the combination of tedizolid and venetoclax targeted the LSC-enriched CD34+CD38- population in ex vivo -cultured primary AML samples. Importantly, normal cord blood hematopoietic stem and progenitor cells were more resistant to this combination suggestive of a wide therapeutic index .To decipher the mechanism by which tedizolid overcomes venetoclax resistance, we first profiled the expression of BCL-2 family members in total cellular and purified mitochondrial extracts. Antibiotic treatment did not affect total BCL-2, MCL-1, or BCL-xL protein levels but caused a substantial increase in BCL-2 levels in the mitochondrial fraction indicative of BCL-2 translocation to the organelle. Next, we confirmed that tedizolid treatment selectively reduced the expression of mitochondrial over nuclear-encoded proteins consistent with its inhibitory effect on mitochondrial translation. This mitonuclear protein imbalance has been shown to activate the integrated stress response (ISR). Tedizolid treatment activated this response as evidenced by an increase in eIF2-α phosphorylation and expression of ATF4, a master transcription factor regulator. To determine whether ISR activation is sufficient to overcome venetoclax resistance, we treated resistant cells with a small-molecule activator of PERK (CCT020312), which activates the ISR through direct eIF2-α phosphorylation, and observed re-sensitization to venetoclax.In summary, our results demonstrate that inhibition of mitochondrial translation, which activates ISR and triggers BCL-2 translocation to the mitochondria, is a novel approach to overcoming venetoclax resistance in AML cells. Our findings provide the rationale for exploring the use protein synthesis inhibitor antibiotics or direct ISR activators in combination with venetoclax for the treatment of AML or other malignancies. DisclosuresNo relevant conflicts of interest to declare.

The Antibiotic Doxycycline Impairs Cardiac Mitochondrial and Contractile Function.

Tetracycline antibiotics act by inhibiting bacterial protein translation. Given the bacterial ancestry of mitochondria, we tested the hypothesis that doxycycline—which belongs to the tetracycline class—reduces mitochondrial function, and results in cardiac contractile dysfunction in cultured H9C2 cardiomyoblasts, adult rat cardiomyocytes, in Drosophila and in mice. Ampicillin and carbenicillin were used as control antibiotics since these do not interfere with mitochondrial translation. In line with its specific inhibitory effect on mitochondrial translation, doxycycline caused a mitonuclear protein imbalance in doxycycline-treated H9C2 cells, reduced maximal mitochondrial respiration, particularly with complex I substrates, and mitochondria appeared fragmented. Flux measurements using stable isotope tracers showed a shift away from OXPHOS towards glycolysis after doxycycline exposure. Cardiac contractility measurements in adult cardiomyocytes and Drosophila melanogaster hearts showed an increased diastolic calcium concentration, and a higher arrhythmicity index. Systolic and diastolic dysfunction were observed after exposure to doxycycline. Mice treated with doxycycline showed mitochondrial complex I dysfunction, reduced OXPHOS capacity and impaired diastolic function. Doxycycline exacerbated diastolic dysfunction and reduced ejection fraction in a diabetes mouse model vulnerable for metabolic derangements. We therefore conclude that doxycycline impairs mitochondrial function and causes cardiac dysfunction.

Exercise alters the mitochondrial proteostasis and induces the mitonuclear imbalance and UPRmt in the hypothalamus of mice

The maintenance of mitochondrial activity in hypothalamic neurons is determinant to the control of energy homeostasis in mammals. Disturbs in the mitochondrial proteostasis can trigger the mitonuclear imbalance and mitochondrial unfolded protein response (UPRmt) to guarantee the mitochondrial integrity and function. However, the role of mitonuclear imbalance and UPRmt in hypothalamic cells are unclear. Combining the transcriptomic analyses from BXD mice database and in vivo experiments, we demonstrated that physical training alters the mitochondrial proteostasis in the hypothalamus of C57BL/6J mice. This physical training elicited the mitonuclear protein imbalance, increasing the mtCO-1/Atp5a ratio, which was accompanied by high levels of UPRmt markers in the hypothalamus. Also, physical training increased the maximum mitochondrial respiratory capacity in the brain. Interestingly, the transcriptomic analysis across several strains of the isogenic BXD mice revealed that hypothalamic mitochondrial DNA-encoded genes were negatively correlated with body weight and several genes related to the orexigenic response. As expected, physical training reduced body weight and food intake. Interestingly, we found an abundance of mt-CO1, a mitochondrial DNA-encoded protein, in NPY-producing neurons in the lateral hypothalamus nucleus of exercised mice. Collectively, our data demonstrated that physical training altered the mitochondrial proteostasis and induced the mitonuclear protein imbalance and UPRmt in hypothalamic cells.

Genome-wide effect of tetracycline, doxycycline and 4-epidoxycycline on gene expression in Saccharomyces cerevisiae.

Tetracycline (Tet) and derivative chemicals (e.g., doxycycline or Dox) have gained widespread recognition for their antibiotic properties since their introduction in the late 1970s, but recent work with these chemicals in the lab has shifted to include multiple techniques in all genetic model systems for the precise control of gene expression. The most widely used Tet‐modulated methodology is the Tet‐On/Tet‐Off gene expression system. Tet is generally considered to have effects specific to bacteria; therefore, it should have few off‐target effects when used in eukaryotic systems, and a previous study in the yeast Saccharomyces cerevisiae found that Dox had no effect on genome‐wide gene expression as measured by microarray. In contrast, another study found that the use of Dox in common cell lines and several model organisms led to mitonuclear protein imbalance, suggesting an inhibitory role of Dox in eukaryotic mitochondria. Recently, a new Dox derivative, 4‐epidoxycycline (4‐ED) was developed that was shown to have less off‐target consequences on mitochondrial health. To determine the best tetracycline family chemical to use for gene expression control in S. cerevisiae, we performed RNA sequencing (RNA‐seq) on yeast grown on standard medium compared with growth on media supplemented with Tet, Dox or 4‐ED. We found each caused dozens of genes to change expression, with Dox eliciting the greatest expression responses, suggesting that the specific tetracycline used in experiments should be tailored to the specific gene(s) of interest when using the Tet‐On/Tet‐Off system to reduce the consequences of confounding off‐target responses.

Oxidative stress induces different tissue dependent effects on Mutyh-deficient mice

8-oxoguanine (8-oxoG) is one of the most prevalent genotoxic lesions, and it is generated in DNA attacked by reactive oxygen species (ROS). Adenine misincorporated opposite to 8-oxoG during replication is excised by MutY homolog (MUTYH), an important protein of the base excision repair (BER) system. Mutyh plays an important role in the maintenance of genomic integrity, but the functional consequences of Mutyh deficiency are not fully understood. In the current study, we investigated the histological and functional changes of five tissues (hippocampus, heart, liver, kidney and lung) and their molecular basis in Mutyh−/− and wild-type mice exposed to D-galactose (D-gal). Our data indicated that Mutyh deficiency hindered the weight gain of experimental mice and induced substantial alterations of 8-oxoG content and superoxide dismutase (SOD) activity, but no significant histological and functional impairment appeared in the investigated tissues of Mutyh- deficient mice without D-gal exposure. Under low-dose D-gal exposure, Mutyh deficiency altered expression of genes involved in mitochondrial unfolded protein response (UPRmt) in the heart, liver and lung, and caused an enhanced expression of mitochondrial dynamics proteins (MDPs) in hippocampus and liver. The stress responses could maintain mitochondrial proteostasis and function. However, such responses were not noted when experiencing excessive damage burden induced by high-dose D-gal exposure, in which Mutyh deficiency increased accumulation of 8-oxoG and aggravated mitonuclear protein imbalance, as well as histological lesions in heart, liver and kidney. A higher sensitivity to ROS-induced cardiotoxicity with high-dose D-gal exposure was noticed in Mutyh−/− mice. However, no differences in learning and memory impairments were observed between Mutyh−/− and wild-type mice with high-dose D-gal exposure. In conclusion, our data demonstrated that Mutyh deficiency has different impacts on various tissues based on the degree of oxidative stress.

Fancd2-deficient hematopoietic stem and progenitor cells depend on augmented mitochondrial translation for survival and proliferation.

Members of the Fanconi anemia (FA) protein family are involved in multiple cellular processes including response to DNA damage and oxidative stress. Here we show that a major FA protein, Fancd2, plays a role in mitochondrial biosynthesis through regulation of mitochondrial translation. Fancd2 interacts with Atad3 and Tufm, which are among the most frequently identified components of the mitochondrial nucleoid complex essential for mitochondrion biosynthesis. Deletion of Fancd2 in mouse hematopoietic stem and progenitor cells (HSPCs) leads to increase in mitochondrial number, and enzyme activity of mitochondrion-encoded respiratory complexes. Fancd2 deficiency increases mitochondrial protein synthesis and induces mitonuclear protein imbalance. Furthermore, Fancd2-deficient HSPCs show increased mitochondrial respiration and mitochondrial reactive oxygen species. By using a cell-free assay with mitochondria isolated from WT and Fancd2-KO HSPCs, we demonstrate that the increased mitochondrial protein synthesis observed in Fancd2-KO HSPCs was directly linked to augmented mitochondrial translation. Finally, Fancd2-deficient HSPCs are selectively sensitive to mitochondrial translation inhibition and depend on augmented mitochondrial translation for survival and proliferation. Collectively, these results suggest that Fancd2 restricts mitochondrial activity through regulation of mitochondrial translation, and that augmented mitochondrial translation and mitochondrial respiration may contribute to HSC defect and bone marrow failure in FA.

IDH1‐mutant cancer cells are sensitive to cisplatin and an IDH1‐mutant inhibitor counteracts this sensitivity

IntroductionIsocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer and the presence of a mutation is associated with improved responses to irradiation and chemotherapy in solid tumor cells. Mutated IDH1 (IDH1MUT) enzymes consume NADPH to produce D‐2‐hydroxyglutarate (D‐2HG) resulting in a decreased reducing power needed for detoxification of e.g. reactive oxygen species (ROS). The objective of the current study was to investigate the mechanism behind the chemosensitivity of the widely‐used anti‐cancer agent cisplatin in IDH1MUT cancer cells.MethodsOxidative stress, DNA damage and mitochondrial dysfunction caused by cisplatin treatment were monitored in IDH1MUT HCT116 cells and U251 glioma cells.ResultsBy performing histo‐ and cytochemical experiments, we found that exposure to cisplatin induced higher levels of ROS, DNA double‐strand breaks (DSBs), and cell death in IDH1MUT cancer cells as compared to IDH1 wild‐type (IDH1WT) cells. Mechanistic investigations as in situ histochemical enzyme activity assays revealed that cisplatin treatment dose‐dependently reduced oxidative respiration in lDH1MUT cells, which was accompanied by disturbed mitochondrial proteostasis, indicative for impaired mitochondrial activity. These effects were abolished by the IDH1MUT inhibitor AGI‐5198 and were recapitulated by treatment with D‐2HG.ConclusionsOur study shows that altered oxidative stress responses and a vulnerable oxidative metabolism underlie the sensitivity of IDH1MUT cancer cells to cisplatin. Our study may have clinical implications and imply that administration of IDH1MUT inhibitors to patients with IDH1MUT cancer abolishes the therapeutic effect of cisplatin. Our in vitro results suggest that concomitant administration of IDH1MUT inhibitors and cisplatin may result in an unfavorable clinical outcome.Support or Funding InformationThis work was supported by Dutch Cancer Society Grants KWF‐UVA 2014‐6839 (to M.K., V.V.V.H., R.J.M., and C.J.F.V.N.) and AMC2016.1‐10460 (to M.K., R.J.M., J.W.W., and C.J.F.V.N.). A) Proliferation assay using HCT116 cells after cisplatin exposure and in the presence or absence of ROS‐scavenging NAC.B) Colony‐forming assay after cisplatin exposure with HCT116 cells in the presence or absence of AGI‐5198 or of D‐2HG.C) Representative photomicrographs and plots of cells in the presence or absence of AGI‐5198, and cisplatin treatment. Y‐H2AX was stained immunocytochemically (red) to demonstrate DNA DSBs and with DAPI (blue) to demonstrate DNA nucleus content.imageA) Proliferation assay using HCT116 cells after cisplatin exposure and in the presence or absence of ROS‐scavenging NAC.B) Colony‐forming assay after cisplatin exposure with HCT116 cells in the presence or absence of AGI‐5198 or of D‐2HG.C) Representative photomicrographs and plots of cells in the presence or absence of AGI‐5198, and cisplatin treatment. Y‐H2AX was stained immunocytochemically (red) to demonstrate DNA DSBs and with DAPI (blue) to demonstrate DNA nucleus content. D) The basal oxygen consumption rate of HCT116 cells shown in the presence or absence of cisplatin. E) Western blot and mitonuclear protein imbalance of MTCO1 and nuclear DNA–encoded SDHA expression in HCT116 cells after exposure to cisplatin or carboplatin. F) Representative monochromatic light photomicrographs and quantification of histochemical enzyme activity of HCT116 cells treated with cisplatin or left untreated after staining for SDH activity in the presence or absence of sodium azide.imageD) The basal oxygen consumption rate of HCT116 cells shown in the presence or absence of cisplatin. E) Western blot and mitonuclear protein imbalance of MTCO1 and nuclear DNA–encoded SDHA expression in HCT116 cells after exposure to cisplatin or carboplatin. F) Representative monochromatic light photomicrographs and quantification of histochemical enzyme activity of HCT116 cells treated with cisplatin or left untreated after staining for SDH activity in the presence or absence of sodium azide.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-382 silencing induces a mitonuclear protein imbalance and activates the mitochondrial unfolded protein response in muscle cells.

Proper mitochondrial function plays a central role in cellular metabolism. Various diseases as well as aging are associated with diminished mitochondrial function. Previously, we identified 19 miRNAs putatively involved in the regulation of mitochondrial metabolism in skeletal muscle, a highly metabolically active tissue. In the current study, these 19 miRNAs were individually silenced in C2C12 myotubes using antisense oligonucleotides, followed by measurement of the expression of 27 genes known to play a major role in regulating mitochondrial metabolism. Based on the outcomes, we then focused on miR‐382‐5p and identified pathways affected by its silencing using microarrays, investigated protein expression, and studied cellular respiration. Silencing of miRNA‐382‐5p significantly increased the expression of several genes involved in mitochondrial dynamics and biogenesis. Conventional microarray analysis in C2C12 myotubes silenced for miRNA‐382‐5p revealed a collective downregulation of mitochondrial ribosomal proteins and respiratory chain proteins. This effect was accompanied by an imbalance between mitochondrial proteins encoded by the nuclear and mitochondrial DNA (1.35‐fold, p < 0.01) and an induction of HSP60 protein (1.31‐fold, p < 0.05), indicating activation of the mitochondrial unfolded protein response (mtUPR). Furthermore, silencing of miR‐382‐5p reduced basal oxygen consumption rate by 14% ( p < 0.05) without affecting mitochondrial content, pointing towards a more efficient mitochondrial function as a result of improved mitochondrial quality control. Taken together, silencing of miR‐382‐5p induces a mitonuclear protein imbalance and activates the mtUPR in skeletal muscle, a phenomenon that was previously associated with improved longevity.

Choline ameliorates cardiac hypertrophy by regulating metabolic remodelling and UPRmt through SIRT3-AMPK pathway.

Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization, but the molecular events underlying the metabolic remodelling remain poorly understood. We explored metabolic remodelling and mitochondrial dysfunction in cardiac hypertrophy and investigated the cardioprotective effects of choline. The experiments were conducted using a model of ventricular hypertrophy by partially banding the abdominal aorta of Sprague Dawley rats. Cardiomyocyte size and cardiac fibrosis were significantly increased in hypertrophic hearts. In vitro cardiomyocyte hypertrophy was induced by exposing neonatal rat cardiomyocytes to angiotensin II (Ang II) (10-6 M, 24 h). Choline attenuated the mito-nuclear protein imbalance and activated the mitochondrial-unfolded protein response (UPRmt) in the heart, thereby preserving the ultrastructure and function of mitochondria in the context of cardiac hypertrophy. Moreover, choline inhibited myocardial metabolic dysfunction by promoting the expression of proteins involved in ketone body and fatty acid metabolism in response to pressure overload, accompanied by the activation of sirtuin 3/AMP-activated protein kinase (SIRT3-AMPK) signalling. In vitro analyses demonstrated that SIRT3 siRNA diminished choline-mediated activation of ketone body metabolism and UPRmt, as well as inhibition of hypertrophic signals. Intriguingly, serum from choline-treated abdominal aorta banding models (where β-hydroxybutyrate was increased) attenuated Ang II-induced myocyte hypertrophy, which indicates that β-hydroxybutyrate is important for the cardioprotective effects of choline. Choline attenuated cardiac dysfunction by modulating the expression of proteins involved in ketone body and fatty acid metabolism, and induction of UPRmt; this was likely mediated by activation of the SIRT3-AMPK pathway. Taken together, these results identify SIRT3-AMPK as a key cardiac transcriptional regulator that helps orchestrate an adaptive metabolic response to cardiac stress. Choline treatment may represent a new therapeutic strategy for optimizing myocardial metabolism in the context of hypertrophy and heart failure.

Systems Phytohormone Responses to Mitochondrial Proteotoxic Stress

Mitochondrial function is controlled by two separate genomes. This feature makes mitochondria prone to proteotoxic stress when a stoichiometric imbalance occurs in the protein complexes that perform oxidative phosphorylation, which consist of both nuclear- and mitochondrial-encoded proteins. Such a proteotoxic stress is known to induce the mitochondrial unfolded protein response (UPRmt) in animals. It is unknown whether UPRmt occurs in plants. Here, we induced a mitonuclear protein imbalance in Arabidopsis through chemical or genetic interference. Mitochondrial proteotoxic stress activated a plant-specific UPRmt and impaired plant growth and development. The plant UPRmt pathway is triggered by a transient oxidative burst, activating MAPK and hormonal (involving ethylene and auxin) signaling, which are all geared to repair proteostasis. This also establishes phytohormones as bona fide plant mitokines. Our data ascertain that mitochondrial protein quality control pathways, such as the UPRmt, are conserved in plants and that hormone signaling is an essential mediator that regulates mitochondrial proteostasis.

Antibiotics induce mitonuclear protein imbalance but fail to inhibit respiration and nutrient activation in pancreatic β-cells

Chloramphenicol and several other antibiotics targeting bacterial ribosomes inhibit mitochondrial protein translation. Inhibition of mitochondrial protein synthesis leads to mitonuclear protein imbalance and reduced respiratory rates as confirmed here in HeLa and PC12 cells. Unexpectedly, respiration in INS-1E insulinoma cells and primary human islets was unaltered in the presence of chloramphenicol. Resting respiratory rates and glucose stimulated acceleration of respiration were also not lowered when a range of antibiotics including, thiamphenicol, streptomycin, gentamycin and doxycycline known to interfere with bacterial protein synthesis were tested. However, chloramphenicol efficiently reduced mitochondrial protein synthesis in INS-1E cells, lowering expression of the mtDNA encoded COX1 subunit of the respiratory chain but not the nuclear encoded ATP-synthase subunit ATP5A. Despite a marked reduction of the essential respiratory chain subunit COX1, normal respiratory rates were maintained in INS-1E cells. ATP-synthase dependent respiration was even elevated in chloramphenicol treated INS-1E cells. Consistent with these findings, glucose-dependent calcium signaling reflecting metabolism-secretion coupling in beta-cells, was augmented. We conclude that antibiotics targeting mitochondria are able to cause mitonuclear protein imbalance in insulin secreting cells. We hypothesize that in contrast to other cell types, compensatory mechanisms are sufficiently strong to maintain normal respiratory rates and surprisingly even result in augmented ATP-synthase dependent respiration and calcium signaling following glucose stimulation. The result suggests that in insulin secreting cells only lowering COX1 below a threshold level may result in a measurable impairment of respiration. When focusing on mitochondrial function, care should be taken when including antibiotics targeting translation for long-term cell culture as depending on the sensitivity of the cell type analyzed, respiration, mitonuclear protein imbalance or down-stream signaling may be altered.

Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice

NAD+ availability decreases with age and in certain disease conditions. Nicotinamide mononucleotide (NMN), a key NAD+ intermediate, has been shown to enhance NAD+ biosynthesis and ameliorate various pathologies in mouse disease models. Inthis study, we conducted a 12-month-long NMN administration to regular chow-fed wild-type C57BL/6N mice during their normal aging. Orally administered NMN was quickly utilized to synthesize NAD+ in tissues. Remarkably, NMN effectively mitigates age-associated physiological decline in mice. Without any obvious toxicity or deleterious effects, NMN suppressed age-associated body weight gain, enhanced energy metabolism, promoted physical activity, improved insulin sensitivity and plasma lipid profile, and ameliorated eye function and other pathophysiologies. Consistent with these phenotypes, NMN prevented age-associated gene expression changes in key metabolic organs and enhanced mitochondrial oxidative metabolism and mitonuclear protein imbalance in skeletal muscle. These effects of NMN highlight the preventive and therapeutic potential of NAD+ intermediates as effective anti-aging interventions in humans.

Disentangling the effect of dietary restriction on mitochondrial function using recombinant inbred mice

Dietary restriction (DR) extends lifespan and healthspan in many species, but precisely how it elicits its beneficial effects is unclear. We investigated the impact of DR on mitochondrial function within liver and skeletal muscle of female ILSXISS mice that exhibit strain-specific variation in lifespan under 40% DR. Strains TejJ89 (lifespan increased under DR), TejJ48 (lifespan unaffected by DR) and TejJ114 (lifespan decreased under DR) were studied following 10 months of 40% DR (13 months of age). Oxygen consumption rates (OCR) within isolated liver mitochondria were unaffected by DR in TejJ89 and TejJ48, but decreased by DR in TejJ114. DR had no effect on hepatic protein levels of PGC-1a, TFAM, and OXPHOS complexes IV. Mitonuclear protein imbalance (nDNA:mtDNA ratio) was unaffected by DR, but HSP90 protein levels were reduced in TejJ114 under DR. Surprisingly hepatic mitochondrial hydrogen peroxide (H2O2) production was elevated by DR in TejJ89, with total superoxide dismutase activity and protein carbonyls increased by DR in both TejJ89 and TejJ114. In skeletal muscle, DR had no effect on mitochondrial OCR, OXPHOS complexes or mitonuclear protein imbalance, but H2O2 production was decreased in TejJ114 and nuclear PGC-1a increased in TejJ89 under DR. Our findings indicate that hepatic mitochondrial dysfunction associated with reduced lifespan of TejJ114 mice under 40% DR, but similar dysfunction was not apparent in skeletal muscle mitochondria. We highlight tissue-specific differences in the mitochondrial response in ILSXISS mice to DR, and underline the importance and challenges of exploiting genetic heterogeneity to help understand mechanisms of ageing.

The Roles of Mutation, Selection, and Expression in Determining Relative Rates of Evolution in Mitochondrial versus Nuclear Genomes.

Eukaryotes rely on proteins encoded by the nuclear and mitochondrial (mt) genomes, which interact within multisubunit complexes such as oxidative-phosphorylation enzymes. Although selection is thought to be less efficient on the asexual mt genome, in bilaterian animals the ratio of nonsynonymous to synonymous substitutions (ω) is lower in mt- compared with nuclear-encoded OXPHOS subunits, suggesting stronger effects of purifying selection in the mt genome. Because high levels of gene expression constrain protein sequence evolution, one proposed resolution to this paradox is that mt genes are expressed more highly than nuclear genes. To test this hypothesis, we investigated expression and sequence evolution of mt and nuclear genes from 84 diverse eukaryotes that vary in mt gene content and mutation rate. We found that the relationship between mt and nuclear ω values varied dramatically across eukaryotes. In contrast, transcript abundance is consistently higher for mt genes than nuclear genes, regardless of which genes happen to be in the mt genome. Consequently, expression levels cannot be responsible for the differences in ω Rather, 84% of the variance in the ratio of ω values between mt and nuclear genes could be explained by differences in mutation rate between the two genomes. We relate these findings to the hypothesis that high rates of mt mutation select for compensatory changes in the nuclear genome. We also propose an explanation for why mt transcripts consistently outnumber their nuclear counterparts, with implications for mitonuclear protein imbalance and aging.