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Mitochondrial fidelity is tightly linked to overall cellular homeostasis and is compromised in ageing and various pathologies 1 – 3 . Mitochondrial malfunction needs to be relayed to the cytosol, where an integrated stress response is triggered by the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) in mammalian cells 4 , 5 . eIF2α phosphorylation is mediated by the four eIF2α kinases GCN2, HRI, PERK and PKR, which are activated by diverse types of cellular stress 6 . However, the machinery that communicates mitochondrial perturbation to the cytosol to trigger the integrated stress response remains unknown 1 , 2 , 7 . Here we combine genome engineering and haploid genetics to unbiasedly identify genes that affect the induction of C/EBP homologous protein (CHOP), a key factor in the integrated stress response. We show that the mitochondrial protease OMA1 and the poorly characterized protein DELE1, together with HRI, constitute the missing pathway that is triggered by mitochondrial stress. Mechanistically, stress-induced activation of OMA1 causes DELE1 to be cleaved into a short form that accumulates in the cytosol, where it binds to and activates HRI via its C-terminal portion. Obstruction of this pathway can be beneficial or adverse depending on the type of mitochondrial perturbation. In addition to the core pathway components, our comparative genetic screening strategy identifies a suite of additional regulators. Together, these findings could be used to inform future strategies to modulate the cellular response to mitochondrial dysfunction in the context of human disease. Haploid genetic screening of cells under different types of mitochondrial perturbation shows that a pathway involving OMA1, DELE1 and the eIF2α kinase HRI communicates mitochondrial stress to the cytosol to trigger the integrated stress response.

Key Points This Review focuses on the pathways that catabolize cellular triacylglycerol ('fat'), namely, neutral lipolysis, acid lipolysis and lipophagy. Neutral lipolysis of triglycerides in cytosolic lipid droplets relies on three lipid hydrolases (lipases): adipose triglyceride lipase, hormone-sensitive lipase and monoacylglycerol lipase. The consecutive action of these enzymes provides free fatty acids and glycerol for energy production and other metabolic pathways during fasting. The regulation of neutral lipolysis is complex and involves numerous proteins, hormones, growth factors and cytokines. Conversely, products and intermediates of neutral lipolysis regulate key metabolic pathways by transcriptional and post-transcriptional mechanisms. Lipophagy is a subtype of macroautophagy. Portions of cytosolic lipid droplets are engulfed by lipoautophagosomes and transported to lysosomes, where triacylglycerols and other lipids undergo acid lipolysis by lysosomal acid lipase. The regulation of acid lipolysis is less complex than the regulation of neutral lipolysis, but again, the products and intermediates of triacylglycerol hydrolysis exit lysosomes and regulate multiple key processes in energy metabolism. Neutral lipolysis, acid lipolysis and lipophagy cooperate mechanistically. Rare mutations in the genes coding for adipose triglyceride lipase; its co-activator, lipid droplet-binding protein CGI-58; hormone-sensitive lipase; and lysosomal acid lipase cause distinct metabolic disorders in humans. Recent insights led to a better understanding of how cellular triacylglycerol catabolism affects the pathogenesis of metabolic diseases, cancer and cancer-associated cachexia and highlighted potential treatment strategies for lipid-associated disorders. Lipolysis degrades triacylglycerols to supply cells with free fatty acids, which are essential components of membrane lipids and substrates for energy production. Recent discoveries transformed our understanding of the functions of and crosstalk between 'neutral' lipolysis, which occurs in the cytosol, and lipophagy and 'acid' lipolysis, which occur in lysosomes, and how dysfunction in these processes contributes to metabolic diseases. Fatty acids are the most efficient substrates for energy production in vertebrates and are essential components of the lipids that form biological membranes. Synthesis of triacylglycerols from non-esterified free fatty acids (FFAs) combined with triacylglycerol storage represents a highly efficient strategy to stockpile FFAs in cells and prevent FFA-induced lipotoxicity. Although essentially all vertebrate cells have some capacity to store and utilize triacylglycerols, white adipose tissue is by far the largest triacylglycerol depot and is uniquely able to supply FFAs to other tissues. The release of FFAs from triacylglycerols requires their enzymatic hydrolysis by a process called lipolysis. Recent discoveries thoroughly altered and extended our understanding of lipolysis. This Review discusses how cytosolic 'neutral' lipolysis and lipophagy, which utilizes 'acid' lipolysis in lysosomes, degrade cellular triacylglycerols as well as how these pathways communicate, how they affect lipid metabolism and energy homeostasis and how their dysfunction affects the pathogenesis of metabolic diseases. Answers to these questions will likely uncover novel strategies for the treatment of prevalent metabolic diseases.

Chronic inflammation is a pathologic feature of neurodegeneration and aging; however, the mechanism regulating this process is not understood. Melatonin, an endogenous free radical scavenger synthesized by neuronal mitochondria, decreases with aging and neurodegeneration. We proposed that insufficient melatonin levels impair mitochondrial homeostasis, resulting in mitochondrial DNA (mtDNA) release and activation of cytosolic DNA-mediated inflammatory response in neurons. We found increased mitochondrial oxidative stress and decreased mitochondrial membrane potential, with higher mtDNA release in brain and primary cerebro-cortical neurons of melatonin-deficient aralkylamine N-acetyltransferase (AANAT) knockout mice. Cytosolic mtDNA activated the cGAS/STING/IRF3 pathway, stimulating inflammatory cytokine generation. We found that Huntington's disease mice had increased mtDNA release, cGAS activation, and inflammation, all inhibited by exogenous melatonin. Thus, we demonstrated that cytosolic mtDNA activated the inflammatory response in aging and neurodegeneration, a process modulated by melatonin. Furthermore, our data suggest that AANAT knockout mice are a model of accelerated aging.

Inflammasomes are multi-protein signaling complexes that trigger the activation of inflammatory caspases and the maturation of interleukin-1β. Among various inflammasome complexes, the NLRP3 inflammasome is best characterized and has been linked with various human autoinflammatory and autoimmune diseases. Thus, the NLRP3 inflammasome may be a promising target for anti-inflammatory therapies. In this review, we summarize the current understanding of the mechanisms by which the NLRP3 inflammasome is activated in the cytosol. We also describe the binding partners of NLRP3 inftammasome complexes activating or inhibiting the inflammasome assembly. Our knowledge of the mechanisms regulating NLRP3 inflammasome signaling and how these influence inflammatory responses offers further insight into potential therapeutic strategies to treat inflammatory diseases associated with dysregulation of the NLRP3 inflammasome,

Sodium chloride promotes vascular fibrosis, arterial hypertension, pro-inflammatory immune cell polarization and endothelial dysfunction, all of which might influence outcomes following stroke. But despite enormous translational relevance, the functional importance of sodium chloride in the pathophysiology of acute ischemic stroke is still unclear. In the current study, we show that high-salt diet leads to significantly worse functional outcomes, increased infarct volumes, and a loss of astrocytes and cortical neurons in acute ischemic stroke. While analyzing the underlying pathologic processes, we identified the migrasome as a novel, sodium chloride-driven pathomechanism in acute ischemic stroke. The migrasome was previously described in vitro as a migrating organelle, which incorporates and dispatches cytosol of surrounding cells and plays a role in intercellular signaling, whereas a pathophysiological meaning has not been elaborated. We here confirm previously reported characteristics of the migrasome in vivo. Immunohistochemistry, electron microscopy and proteomic analyses further demonstrate that the migrasome incorporates and dispatches cytosol of surrounding neurons following stroke. The clinical relevance of these findings is emphasized by neuropathological examinations, which detected migrasome formation in infarcted brain parenchyma of human stroke patients. In summary, we demonstrate that high-salt diet aggravates stroke outcomes, and we characterize the migrasome as a novel mechanism in acute stroke pathophysiology.

Group A Streptococcus (Streptococcus pyogenes or GAS) causes pharyngitis, severe invasive infections, and the post-infectious syndromes of glomerulonephritis and rheumatic fever. GAS can be internalized and killed by epithelial cells in vitro, a process that may contribute to local innate defense against pharyngeal infection. Secretion of the pore-forming toxin streptolysin O (SLO) by GAS has been reported to stimulate targeted autophagy (xenophagy) upon internalization of the bacteria by epithelial cells. Whereas this process was associated with killing of GAS in HeLa cells, studies in human keratinocytes found SLO production enhanced intracellular survival. To reconcile these conflicting observations, we now report in-depth investigation of xenophagy in response to GAS infection of human oropharyngeal keratinocytes, the predominant cell type of the pharyngeal epithelium. We found that SLO expression was associated with prolonged intracellular survival; unexpectedly, expression of the co-toxin NADase was required for this effect. Enhanced intracellular survival was lost upon deletion of NADase or inactivation of its enzymatic activity. Shortly after internalization of GAS by keratinocytes, SLO-mediated damage to the bacteria-containing vacuole resulted in exposure to the cytosol, ubiquitination of GAS and/or associated vacuolar membrane remnants, and engulfment of GAS in LC3-positive vacuoles. We also found that production of streptolysin S could mediate targeting of GAS to autophagosomes in the absence of SLO, a process accompanied by galectin 8 binding to damaged GAS-containing endosomes. Maturation of GAS-containing autophagosome-like vacuoles to degradative autolysosomes was prevented by SLO pore-formation and by SLO-mediated translocation of enzymatically active NADase into the keratinocyte cytosol. We conclude that SLO stimulates xenophagy in pharyngeal keratinocytes, but the coordinated action of SLO and NADase prevent maturation of GAS-containing autophagosomes, thereby prolonging GAS intracellular survival. This novel activity of NADase to block autophagic killing of GAS in pharyngeal cells may contribute to pharyngitis treatment failure, relapse, and chronic carriage.

Motor neuron disease (MND) is a progressive neurodegenerative disease with no effective treatment. One of the principal pathological hallmarks is the deposition of TAR DNA binding protein 43 (TDP-43) in cytoplasmic inclusions. TDP-43 aggregation occurs in both familial and sporadic MND; however, the mechanism of endogenous TDP-43 aggregation in disease is incompletely understood. This study focused on the induction of cytoplasmic accumulation of endogenous TDP-43 in the motor neuronal cell line NSC-34. The endoplasmic reticulum (ER) stressor tunicamycin induced casein kinase 1 (CK1)-dependent cytoplasmic accumulation of endogenous TDP-43 in differentiated NSC-34 cells, as seen by immunocytochemistry. Immunoblotting showed that induction of ER stress had no effect on abundance of TDP-43 or phosphorylated TDP-43 in the NP-40/RIPA soluble fraction. However, there were significant increases in abundance of TDP-43 and phosphorylated TDP-43 in the NP-40/RIPA-insoluble, urea-soluble fraction, including high molecular weight species. In all cases, these increases were lowered by CK1 inhibition. Thus ER stress signalling, as induced by tunicamycin, causes CK1-dependent phosphorylation of TDP-43 and its consequent cytosolic accumulation.

Primary liver cancer comprises a diverse group of liver tumors. The heterogeneity of these tumors is seen as one of the obstacles to finding an effective therapy. The Hippo pathway, with its downstream transcriptional co-activator Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), has a decisive role in the carcinogenesis of primary liver cancer. Therefore, we examined the expression pattern of YAP and TAZ in 141 patients with hepatocellular carcinoma keratin 19 positive (HCC K19+), hepatocellular carcinoma keratin 19 negative (HCC K19−), combined hepatocellular–cholangiocarcinoma carcinoma (cHCC-CCA), or cholangiocarcinoma (CCA). All cHCC-CCA and CCA patients showed high expression levels for YAP and TAZ, while only some patients of the HCC group were positive. Notably, we found that a histoscore of both markers is useful in the challenging diagnosis of cHCC-CCA. In addition, positivity for YAP and TAZ was observed in the hepatocellular and cholangiocellular components of cHCC-CCA, which suggests a single cell origin in cHCC-CCA. Within the K19− HCC group, our results demonstrate that the expression of YAP is a statistically significant predictor of poor prognosis when observed in the cytoplasm. Nuclear expression of TAZ is an even more specific and independent predictor of poor disease-free survival and overall survival of K19− HCC patients. Our results thus identify different levels of YAP/TAZ expression in various liver cancers that can be used for diagnostics.

Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate–aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.

Oxidative stress modulates carcinogenesis in the liver; however, direct evidence for metabolic control of oxidative stress during pathogenesis, particularly, of progression from cirrhosis to hepatocellular carcinoma (HCC), has been lacking. Deficiency of transaldolase (TAL), a rate-limiting enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP), restricts growth and predisposes to cirrhosis and HCC in mice and humans. Here, we show that mitochondrial oxidative stress and progression from cirrhosis to HCC and acetaminophen-induced liver necrosis are critically dependent on NADPH depletion and polyol buildup by aldose reductase (AR), while this enzyme protects from carbon trapping in the PPP and growth restriction in TAL deficiency. Both TAL and AR are confined to the cytosol; however, their inactivation distorts mitochondrial redox homeostasis in opposite directions. The results suggest that AR acts as a rheostat of carbon recycling and NADPH output of the PPP with broad implications for disease progression from cirrhosis to HCC. In this study, Oaks and Patel et al. characterize the crosstalk between the pentose phosphate pathway and mitochondrial redox homeostasis in the context of aldose reductase and transaldolase deficiency and the contribution of pentose phosphate pathway mitochondria deregulation to the progression from cirrhosis to hepatocellular carcinoma.