In a screen designed to identify novel inducers of autophagy, we discovered that STAT3 inhibitors potently stimulate the autophagic flux. Accordingly, genetic inhibition of STAT3 stimulated autophagy in vitro and in vivo, while overexpression of STAT3 variants, encompassing wild-type, nonphosphorylatable, and extranuclear STAT3, inhibited starvation-induced autophagy. The SH2 domain of STAT3 was found to interact with the catalytic domain of the eIF2α kinase 2 EIF2AK2, best known as protein kinase R (PKR). Pharmacological and genetic inhibition of STAT3 stimulated the activating phosphorylation of PKR and consequent eIF2α hyperphosphorylation. Moreover, PKR depletion inhibited autophagy as initiated by chemical STAT3 inhibitors or free fatty acids like palmitate. STAT3-targeting chemicals and palmitate caused the disruption of inhibitory STAT3-PKR interactions, followed by PKR-dependent eIF2α phosphorylation, which facilitates autophagy induction. These results unravel an unsuspected mechanism of autophagy control that involves STAT3 and PKR as interacting partners.

Reduced supply of the amino acid methionine increases longevity across species through an as yet elusive mechanism. Here, we report that methionine restriction (MetR) extends yeast chronological lifespan in an autophagy-dependent manner. Single deletion of several genes essential for autophagy (ATG5, ATG7 or ATG8) fully abolished the longevity-enhancing capacity of MetR. While pharmacological or genetic inhibition of TOR1 increased lifespan in methionine-prototroph yeast, TOR1 suppression failed to extend the longevity of methionine-restricted yeast cells. Notably, vacuole-acidity was specifically enhanced by MetR, a phenotype that essentially required autophagy. Overexpression of vacuolar ATPase components (Vma1p or Vph2p) suffices to increase chronological lifespan of methionine-prototrophic yeast. In contrast, lifespan extension upon MetR was prevented by inhibition of vacuolar acidity upon disruption of the vacuolar ATPase. In conclusion, autophagy promotes lifespan extension upon MetR and requires the subsequent stimulation of vacuolar acidification, while it is epistatic to the equally autophagy-dependent anti-aging pathway triggered by TOR1 inhibition or deletion.

Experimental and bioinformatic studies of transcription initiation by RNA polymerase II (RNAP2) have revealed a mechanism of RNAP2 transcription initiation less uniform across gene promoters than initially thought. However, the general transcription factor TFIIB is presumed to be universally required for RNAP2 transcription initiation. Based on bioinformatic analysis of data and effects of TFIIB knockdown in primary and transformed cell lines on cellular functionality and global gene expression, we report that TFIIB is dispensable for transcription of many human promoters, but is essential for herpes simplex virus-1 (HSV-1) gene transcription and replication. We report a novel cell cycle TFIIB regulation and localization of the acetylated TFIIB variant on the transcriptionally silent mitotic chromatids. Taken together, these results establish a new paradigm for TFIIB functionality in human gene expression, which when downregulated has potent anti-viral effects.

In the course of mitochondrial diseases standard care mostly focuses on treatment of symptoms, while therapeutic approaches aimed at restoring mitochondrial function are currently still in development. The transfer of healthy or modified mitochondria into host cells would open up the possibilities of new cell therapies. Therefore, in this study, a novel method of mitochondrial transfer is proposed by anti-TOM22 magnetic bead-labeled mitochondria with the assistance of a magnetic plate. In comparison to the passive transfer method, the magnetomitotransfer method was more efficient at transferring mitochondria into cells (78-92% vs 0-17% over 3 days). This transfer was also more rapid, with a high ratio of magnetomitotransferred cells and high density of transferred mitochondria within the first day of culture. Importantly, transferred mitochondria appeared to be functional as they strongly enhanced respiration in magnetomitotransferred cells. The novel method of magnetomitotransfer may offer potential for therapeutic approaches for treatment of a variety of mitochondria-associated pathologies, e.g. various neurodegenerative diseases.

Colorectal carcinoma represents a heterogeneous entity, with only a fraction of the tumours responding to available therapies, requiring a better molecular understanding of the disease in precision oncology. To address this challenge, the OncoTrack consortium recruited 106 CRC patients (stages I-IV) and developed a pre-clinical platform generating a compendium of drug sensitivity data totalling >4,000 assays testing 16 clinical drugs on patient-derived in vivo and in vitro models. This large biobank of 106 tumours, 35 organoids and 59 xenografts, with extensive omics data comparing donor tumours and derived models provides a resource for advancing our understanding of CRC. Models recapitulate many of the genetic and transcriptomic features of the donors, but defined less complex molecular sub-groups because of the loss of human stroma. Linking molecular profiles with drug sensitivity patterns identifies novel biomarkers, including a signature outperforming RAS/RAF mutations in predicting sensitivity to the EGFR inhibitor cetuximab.

The steroid hormone progesterone is not only a crucial sex hormone, but also serves as a neurosteroid, thus playing an important role in brain function. Epidemiological data suggest that progesterone improves the recovery of patients after traumatic brain injury. Brain injuries are often connected to elevated calcium spikes, reactive oxygen species (ROS) and programmed cell death affecting neurons. Here, we establish a yeast model to study progesterone-mediated cytoprotection. External supply of progesterone protected yeast cells from apoptosis-inducing stress stimuli and resulted in elevated mitochondrial oxygen uptake accompanied by a drop in ROS generation and ATP levels during chronological aging. In addition, cellular Ca2+ concentrations were reduced upon progesterone treatment, and this effect occurred independently of known Ca2+ transporters and mitochondrial respiration. All effects were also independent of Dap1, the yeast orthologue of the progesterone receptor. Altogether, our observations provide new insights into the cytoprotective effects of progesterone.

Reprimo (RPRM), a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb), RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH) and fluorescent in situ hybridization (FISH), we demonstrate that rprm (rprma/rprmb) and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb) and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS). We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC) staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

We have isolated a microsporidium from a laboratory stock of the European grape vine moth, Lobesia botrana Den. et Schiff. (Lepidoptera, Tortricidae). Screening of this stock showed an infection rate of more than 90%, whereas field collected larvae from three different locations in Rhineland-Palatinate (Germany) did not demonstrate any signs of infection. Light and electron microscopic investigations of infected insects showed that gross pathology, morphology, and ultrastructure of the microsporidium are similar to those described earlier for Pleistophora legeri. Comparative phylogenetic analysis of the small subunit rDNA using maximum likelihood, maximum parsimony, and neighbour joining distance methods showed that our isolate was closely related to Cystosporogenes operophterae. Based on our morphological and molecular investigations we propose to rename this species Cystosporogenes legeri nov. comb.

In yeast, apoptotic cell death can be triggered by various factors such as H2O2, cell aging, or acetic acid. Yeast caspase (Yca1p) and cellular reactive oxygen species (ROS) are key regulators of this process. Here, we show that moderate doses of three virally encoded killer toxins (K1, K28, and zygocin) induce an apoptotic yeast cell response, although all three toxins differ significantly in their primary killing mechanisms. In contrast, high toxin concentrations prevent the occurrence of an apoptotic cell response and rather cause necrotic, toxin-specific cell killing. Studies with Deltayca1 and Deltagsh1 deletion mutants indicate that ROS accumulation as well as the presence of yeast caspase 1 is needed for apoptosis in toxin-treated yeast cells. We conclude that in the natural environment of toxin-secreting killer yeasts, where toxin concentration is usually low, induction of apoptosis might play an important role in efficient toxin-mediated cell killing.

Quiescence is the most common and, arguably, most poorly understood cell cycle state. This is in part because pure populations of quiescent cells are typically difficult to isolate. We report the isolation and characterization of quiescent and nonquiescent cells from stationary-phase (SP) yeast cultures by density-gradient centrifugation. Quiescent cells are dense, unbudded daughter cells formed after glucose exhaustion. They synchronously reenter the mitotic cell cycle, suggesting that they are in a G(0) state. Nonquiescent cells are less dense, heterogeneous, and composed of replicatively older, asynchronous cells that rapidly lose the ability to reproduce. Microscopic and flow cytometric analysis revealed that nonquiescent cells accumulate more reactive oxygen species than quiescent cells, and over 21 d, about half exhibit signs of apoptosis and necrosis. The ability to isolate both quiescent and nonquiescent yeast cells from SP cultures provides a novel, tractable experimental system for studies of quiescence, chronological and replicative aging, apoptosis, and the cell cycle.

Patients with hepatocellular carcinoma (HCC) displaying overexpression of immune gene signatures are likely to be more sensitive to immunotherapy, however, the use of such signatures in clinical settings remains challenging. We thus aimed, using artificial intelligence (AI) on whole-slide digital histological images, to develop models able to predict the activation of 6 immune gene signatures.

The proteolytic turnover of mitochondrial proteins is poorly understood. Here, we used a combination of dynamic isotope labeling and mass spectrometry to gain a global overview of mitochondrial protein turnover in yeast cells. Intriguingly, we found an exceptionally high turnover of the NADH dehydrogenase, Nde1. This homolog of the mammalian apoptosis inducing factor, AIF, forms two distinct topomers in mitochondria, one residing in the intermembrane space while the other spans the outer membrane and is exposed to the cytosol. The surface-exposed topomer triggers cell death in response to pro-apoptotic stimuli. The surface-exposed topomer is degraded by the cytosolic proteasome/Cdc48 system and the mitochondrial protease Yme1; however, it is strongly enriched in respiratory-deficient cells. Our data suggest that in addition to their role in electron transfer, mitochondrial NADH dehydrogenases such as Nde1 or AIF integrate signals from energy metabolism and cytosolic proteostasis to eliminate compromised cells from growing populations.

Salicylate, the active derivative of aspirin (acetylsalicylate), recapitulates the mode of action of caloric restriction inasmuch as it stimulates autophagy through the inhibition of the acetyltransferase activity of EP300. Here, we directly compared the metabolic effects of aspirin medication with those elicited by 48 h fasting in mice, revealing convergent alterations in the plasma and the heart metabolome. Aspirin caused a transient reduction of general protein acetylation in blood leukocytes, accompanied by the induction of autophagy. However, these effects on global protein acetylation could not be attributed to the mere inhibition of EP300, as determined by epistatic experiments and exploration of the acetyl-proteome from salicylate-treated EP300-deficient cells. Aspirin reduced high-fat diet-induced obesity, diabetes, and hepatosteatosis. These aspirin effects were observed in autophagy-competent mice but not in two different models of genetic (Atg4b-/- or Bcln1+/-) autophagy-deficiency. Aspirin also improved tumor control by immunogenic chemotherapeutics, and this effect was lost in T cell-deficient mice, as well as upon knockdown of an essential autophagy gene (Atg5) in cancer cells. Hence, the health-improving effects of aspirin depend on autophagy.

A number of natural plant products have a long-standing history in both traditional and modern medical applications. Some secondary metabolites induce autophagy and mediate autophagy-dependent healthspan- and lifespan-extending effects in suitable mouse models. Here, we identified isobacachalcone (ISO) as a non-toxic inducer of autophagic flux that acts on human and mouse cells in vitro, as well as mouse organs in vivo. Mechanistically, ISO inhibits AKT as well as, downstream of AKT, the mechanistic target of rapamycin complex 1 (mTORC1), coupled to the activation of the pro-autophagic transcription factors EB (TFEB) and E3 (TFE3). Cells equipped with a constitutively active AKT mutant failed to activate autophagy. ISO also stimulated the AKT-repressible activation of all three arms of the unfolded stress response (UPR), including the PERK-dependent phosphorylation of eukaryotic initiation factor 2α (eIF2α). Knockout of TFEB and/or TFE3 blunted the UPR, while knockout of PERK or replacement of eIF2α by a non-phosphorylable mutant reduced TFEB/TFE3 activation and autophagy induced by ISO. This points to crosstalk between the UPR and autophagy. Of note, the administration of ISO to mice improved the efficacy of immunogenic anticancer chemotherapy. This effect relied on an improved T lymphocyte-dependent anticancer immune response and was lost upon constitutive AKT activation in, or deletion of the essential autophagy gene Atg5 from, the malignant cells. In conclusion, ISO is a bioavailable autophagy inducer that warrants further preclinical characterization.

Life and health span can be prolonged by calorie limitation or by pharmacologic agents that mimic the effects of caloric restriction. Both starvation and the genetic inactivation of nutrient signaling converge on the induction of autophagy, a cytoplasmic recycling process that counteracts the age-associated accumulation of damaged organelles and proteins as it improves the metabolic fitness of cells. Here we review experimental findings indicating that inhibition of the major nutrient and growth-related signaling pathways as well as the upregulation of anti-aging pathways mediate life span extension via the induction of autophagy. Furthermore, we discuss mounting evidence suggesting that autophagy is not only necessary but, at least in some cases, also sufficient for increasing longevity.

Dietary restriction (DR) without malnutrition encompasses numerous regimens with overlapping benefits including longevity and stress resistance, but unifying nutritional and molecular mechanisms remain elusive. In a mouse model of DR-mediated stress resistance, we found that sulfur amino acid (SAA) restriction increased expression of the transsulfuration pathway (TSP) enzyme cystathionine γ-lyase (CGL), resulting in increased hydrogen sulfide (H2S) production and protection from hepatic ischemia reperfusion injury. SAA supplementation, mTORC1 activation, or chemical/genetic CGL inhibition reduced H2S production and blocked DR-mediated stress resistance. In vitro, the mitochondrial protein SQR was required for H2S-mediated protection during nutrient/oxygen deprivation. Finally, TSP-dependent H2S production was observed in yeast, worm, fruit fly, and rodent models of DR-mediated longevity. Together, these data are consistent with evolutionary conservation of TSP-mediated H2S as a mediator of DR benefits with broad implications for clinical translation. PAPERFLICK:

Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.

Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1).

To identify new genetic regulators of cellular aging and senescence, we performed genome-wide comparative RNA profiling with selected human cellular model systems, reflecting replicative senescence, stress-induced premature senescence, and distinct other forms of cellular aging. Gene expression profiles were measured, analyzed, and entered into a newly generated database referred to as the GiSAO database. Bioinformatic analysis revealed a set of new candidate genes, conserved across the majority of the cellular aging models, which were so far not associated with cellular aging, and highlighted several new pathways that potentially play a role in cellular aging. Several candidate genes obtained through this analysis have been confirmed by functional experiments, thereby validating the experimental approach. The effect of genetic deletion on chronological lifespan in yeast was assessed for 93 genes where (i) functional homologues were found in the yeast genome and (ii) the deletion strain was viable. We identified several genes whose deletion led to significant changes of chronological lifespan in yeast, featuring both lifespan shortening and lifespan extension. In conclusion, an unbiased screen across species uncovered several so far unrecognized molecular pathways for cellular aging that are conserved in evolution.

Pathological neuronal inclusions of the 43-kDa TAR DNA-binding protein (TDP-43) are implicated in dementia and motor neuron disorders; however, the molecular mechanisms of the underlying cell loss remain poorly understood. Here we used a yeast model to elucidate cell death mechanisms upon expression of human TDP-43. TDP-43-expressing cells displayed markedly increased markers of oxidative stress, apoptosis, and necrosis. Cytotoxicity was dose- and age-dependent and was potentiated upon expression of disease-associated variants. TDP-43 was localized in perimitochondrial aggregate-like foci, which correlated with cytotoxicity. Although the deleterious effects of TDP-43 were significantly decreased in cells lacking functional mitochondria, cell death depended neither on the mitochondrial cell death proteins apoptosis-inducing factor, endonuclease G, and cytochrome c nor on the activity of cell death proteases like the yeast caspase 1. In contrast, impairment of the respiratory chain attenuated the lethality upon TDP-43 expression with a stringent correlation between cytotoxicity and the degree of respiratory capacity or mitochondrial DNA stability. Consistently, an increase in the respiratory capacity of yeast resulted in enhanced TDP-43-triggered cytotoxicity, oxidative stress, and cell death markers. These data demonstrate that mitochondria and oxidative stress are important to TDP-43-triggered cell death in yeast and may suggest a similar role in human TDP-43 pathologies.