Global, Regional, and National Economic Consequences of Stroke.

BACKGROUND: An understanding of global, regional, and national macroeconomic losses caused by stroke is important for allocation of clinical and research resources. The authors investigated the macroeconomic consequences of stroke disease burden in the year 2019 in 173 countries. METHODS: Disability-adjusted life year data for overall stroke and its subtypes (ischemic stroke, intracerebral hemorrhage, and subarachnoid hemorrhage) were collected from the GBD study (Global Burden of Disease) 2019 database. Gross domestic product (GDP, adjusted for purchasing power parity [PPP]) data were collected from the World Bank; GDP and disability-adjusted life year data were combined to estimate macroeconomic losses using a value of lost welfare (VLW) approach. All results are presented in 2017 international US dollars adjusted for PPP. RESULTS: Globally, in 2019, VLW due to stroke was $2059.67 billion or 1.66% of the global GDP. Global VLW/GDP for stroke subtypes was 0.78% (VLW=$964.51 billion) for ischemic stroke, 0.71% (VLW=$882.81 billion) for intracerebral hemorrhage, and 0.17% (VLW=$212.36 billion) for subarachnoid hemorrhage. The Central European, Eastern European, and Central Asian GBD super-region reported the highest VLW/GDP for stroke overall (3.01%), ischemic stroke (1.86%), and for subarachnoid hemorrhage (0.26%). The Southeast Asian, East Asian, and Oceanian GBD super-region reported the highest VLW/GDP for intracerebral hemorrhage (1.48%). CONCLUSIONS: The global macroeconomic consequences related to stroke are vast even when considering stroke subtypes. The present quantification may be leveraged to help justify increased spending of finite resources on stroke in an effort to improve outcomes for patients with stroke globally.

Integrative transcriptomic and metabolic analyses of the mammalian hibernating brain identifies a key role for succinate dehydrogenase in ischemic tolerance.

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites 1 . Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus , provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage 2 . Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and α-ketoglutarate-αKG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo . Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

Early Neutrophilia Marked by Aerobic Glycolysis Sustains Host Metabolism and Delays Cancer Cachexia.

An elevated neutrophil-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, using murine model systems of colorectal and pancreatic cancer we show that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, similar to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening the overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution, and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.

A Novel Combination of Blood Biomarkers and Clinical Stroke Scales Facilitates Detection of Large Vessel Occlusion Ischemic Strokes.

Acute ischemic stroke caused by large vessel occlusions (LVOs) is a major contributor to stroke deaths and disabilities; however, identification for emergency treatment is challenging. We recruited two separate cohorts of suspected stroke patients and screened a panel of blood-derived protein biomarkers for LVO detection. Diagnostic performance was estimated by using blood biomarkers in combination with NIHSS-derived stroke severity scales. Multivariable analysis demonstrated that D-dimer (OR 16, 95% CI 5-60; p-value < 0.001) and GFAP (OR 0.002, 95% CI 0-0.68; p-value < 0.05) comprised the optimal panel for LVO detection. Combinations of D-dimer and GFAP with a number of stroke severity scales increased the number of true positives, while reducing false positives due to hemorrhage, as compared to stroke scales alone (p-value < 0.001). A combination of the biomarkers with FAST-ED resulted in the highest accuracy at 95% (95% CI: 87-99%), with sensitivity of 91% (95% CI: 72-99%), and specificity of 96% (95% CI: 90-99%). Diagnostic accuracy was confirmed in an independent cohort, in which accuracy was again shown to be 95% (95% CI: 87-99%), with a sensitivity of 82% (95% CI: 57-96%), and specificity of 98% (95% CI: 92-100%). Accordingly, the combination of D-dimer and GFAP with stroke scales may provide a simple and highly accurate tool for identifying LVO patients, with a potential impact on time to treatment.

Transcriptomic analysis of human primary breast cancer identifies fatty acid oxidation as a target for metformin.

BACKGROUND: Epidemiological studies suggest that metformin may reduce the incidence of cancer in patients with diabetes and multiple late phase clinical trials assessing the potential of repurposing this drug are underway. Transcriptomic profiling of tumour samples is an excellent tool to understand drug bioactivity, identify candidate biomarkers and assess for mechanisms of resistance to therapy. METHODS: Thirty-six patients with untreated primary breast cancer were recruited to a window study and transcriptomic profiling of tumour samples carried out before and after metformin treatment. RESULTS: Multiple genes that regulate fatty acid oxidation were upregulated at the transcriptomic level and there was a differential change in expression between two previously identified cohorts of patients with distinct metabolic responses. Increase in expression of a mitochondrial fatty oxidation gene composite signature correlated with change in a proliferation gene signature. In vitro assays showed that, in contrast to previous studies in models of normal cells, metformin reduces fatty acid oxidation with a subsequent accumulation of intracellular triglyceride, independent of AMPK activation. CONCLUSIONS: We propose that metformin at clinical doses targets fatty acid oxidation in cancer cells with implications for patient selection and drug combinations. CLINICAL TRIAL REGISTRATION: NCT01266486.

Targeted breath analysis: exogenous volatile organic compounds (EVOC) as metabolic pathway-specific probes.

Breath research has almost invariably focussed on the identification of endogenous volatile organic compounds (VOCs) as disease biomarkers. After five decades, a very limited number of breath tests measuring endogenous VOCs is applied to the clinic. In this perspective article, we explore some of the factors that may have contributed to the current lack of clinical applications of breath endogenous VOCs. We discuss potential pitfalls of experimental design, analytical challenges, as well as considerations regarding the biochemical pathways that may impinge on the application of endogenous VOCs as specific disease biomarkers. We point towards several lines of evidence showing that breath analysis based on administration of exogenous compounds has been a more successful strategy, with several tests currently applied to the clinic, compared to measurement of endogenous VOCs. Finally, we propose a novel approach, based on the use of exogenous VOC (EVOC) probes as potential strategy to measure the activity of metabolic enzymes in vivo, as well as the function of organs, through breath analysis. We present longitudinal data showing the potential of EVOC probe strategies in breath analysis. We also gathered important data showing that administration of EVOC probes induces significant changes compared to previous exposures to the same compounds. EVOC strategies could herald a new wave of substrate-based breath tests, potentially bridging the gap between research tools and clinical applications.

Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo.

Mutations of the mitochondrial genome (mtDNA) underlie a substantial portion of mitochondrial disease burden. These disorders are currently incurable and effectively untreatable, with heterogeneous penetrance, presentation and prognosis. To address the lack of effective treatment for these disorders, we exploited a recently developed mouse model that recapitulates common molecular features of heteroplasmic mtDNA disease in cardiac tissue: the m.5024C>T tRNAAla mouse. Through application of a programmable nuclease therapy approach, using systemically administered, mitochondrially targeted zinc-finger nucleases (mtZFN) delivered by adeno-associated virus, we induced specific elimination of mutant mtDNA across the heart, coupled to a reversion of molecular and biochemical phenotypes. These findings constitute proof of principle that mtDNA heteroplasmy correction using programmable nucleases could provide a therapeutic route for heteroplasmic mitochondrial diseases of diverse genetic origin.

Integrated Pharmacodynamic Analysis Identifies Two Metabolic Adaption Pathways to Metformin in Breast Cancer.

Late-phase clinical trials investigating metformin as a cancer therapy are underway. However, there remains controversy as to the mode of action of metformin in tumors at clinical doses. We conducted a clinical study integrating measurement of markers of systemic metabolism, dynamic FDG-PET-CT, transcriptomics, and metabolomics at paired time points to profile the bioactivity of metformin in primary breast cancer. We show metformin reduces the levels of mitochondrial metabolites, activates multiple mitochondrial metabolic pathways, and increases 18-FDG flux in tumors. Two tumor groups are identified with distinct metabolic responses, an OXPHOS transcriptional response (OTR) group for which there is an increase in OXPHOS gene transcription and an FDG response group with increased 18-FDG uptake. Increase in proliferation, as measured by a validated proliferation signature, suggested that patients in the OTR group were resistant to metformin treatment. We conclude that mitochondrial response to metformin in primary breast cancer may define anti-tumor effect.

Breath biopsy for early detection and precision medicine in cancer.

Breath biopsy enables the non-invasive collection and analysis of volatile organic compounds (VOCs) in exhaled breath, providing valuable information about disease processes occurring in the body. Metabolic changes occur in cancer cells at the earliest stages of disease. We discuss progress in the use of breath biopsy for discovery of breath-based biomarkers for early detection of cancer, and potential applications for breath biopsy in enabling precision medicine in cancer.

mTORC1 Upregulation Leads to Accumulation of the Oncometabolite Fumarate in a Mouse Model of Renal Cell Carcinoma.

Renal cell carcinomas (RCCs) are common cancers diagnosed in more than 350,000 people each year worldwide. Several pathways are de-regulated in RCCs, including mTORC1. However, how mTOR drives tumorigenesis in this context is unknown. The lack of faithful animal models has limited progress in understanding and targeting RCCs. Here, we generated a mouse model harboring the kidney-specific inactivation of Tsc1. These animals develop cysts that evolve into papillae, cystadenomas, and papillary carcinomas. Global profiling confirmed several metabolic derangements previously attributed to mTORC1. Notably, Tsc1 inactivation results in the accumulation of fumarate and in mTOR-dependent downregulation of the TCA cycle enzyme fumarate hydratase (FH). The re-expression of FH in cellular systems lacking Tsc1 partially rescued renal epithelial transformation. Importantly, the mTORC1-FH axis is likely conserved in human RCC specimens. We reveal a role of mTORC1 in renal tumorigenesis, which depends on the oncometabolite fumarate.

NADH Shuttling Couples Cytosolic Reductive Carboxylation of Glutamine with Glycolysis in Cells with Mitochondrial Dysfunction.

The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.

Mammalian Circadian Period, But Not Phase and Amplitude, Is Robust Against Redox and Metabolic Perturbations.

AIMS: Circadian rhythms permeate all levels of biology to temporally regulate cell and whole-body physiology, although the cell-autonomous mechanism that confers ∼24-h periodicity is incompletely understood. Reports describing circadian oscillations of over-oxidized peroxiredoxin abundance have suggested that redox signaling plays an important role in the timekeeping mechanism. Here, we tested the functional contribution that redox state and primary metabolism make to mammalian cellular timekeeping. RESULTS: We found a circadian rhythm in flux through primary glucose metabolic pathways, indicating rhythmic NAD(P)H production. Using pharmacological and genetic perturbations, however, we found that timekeeping was insensitive to changes in glycolytic flux, whereas oxidative pentose phosphate pathway (PPP) inhibition and other chronic redox stressors primarily affected circadian gene expression amplitude, not periodicity. Finally, acute changes in redox state decreased PER2 protein stability, phase dependently, to alter the subsequent phase of oscillation. INNOVATION: Circadian rhythms in primary cellular metabolism and redox state have been proposed to play a role in the cellular timekeeping mechanism. We present experimental data testing that hypothesis. CONCLUSION: Circadian flux through primary metabolism is cell autonomous, driving rhythmic NAD(P)+ redox cofactor turnover and maintaining a redox balance that is permissive for circadian gene expression cycles. Redox homeostasis and PPP flux, but not glycolysis, are necessary to maintain clock amplitude, but neither redox nor glucose metabolism determines circadian period. Furthermore, cellular rhythms are sensitive to acute changes in redox balance, at least partly through regulation of PER protein. Redox and metabolic state are, thus, both inputs and outputs, but not state variables, of cellular circadian timekeeping. Antioxid. Redox Signal. 28, 507-520.

Fumarate Hydratase Loss Causes Combined Respiratory Chain Defects.

Fumarate hydratase (FH) is an enzyme of the tricarboxylic acid (TCA) cycle mutated in hereditary and sporadic cancers. Despite recent advances in understanding its role in tumorigenesis, the effects of FH loss on mitochondrial metabolism are still unclear. Here, we used mouse and human cell lines to assess mitochondrial function of FH-deficient cells. We found that human and mouse FH-deficient cells exhibit decreased respiration, accompanied by a varying degree of dysfunction of respiratory chain (RC) complex I and II. Moreover, we show that fumarate induces succination of key components of the iron-sulfur cluster biogenesis family of proteins, leading to defects in the biogenesis of iron-sulfur clusters that affect complex I function. We also demonstrate that suppression of complex II activity is caused by product inhibition due to fumarate accumulation. Overall, our work provides evidence that the loss of a single TCA cycle enzyme is sufficient to cause combined RC activity dysfunction.

Mitochondrial Metabolism: Yin and Yang for Tumor Progression.

Altered metabolism is a distinct feature of cancer cells. During transformation, the entire metabolic network is rewired to efficiently convert nutrients to biosynthetic precursors to sustain cancer cell growth and proliferation. Whilst the molecular underpinnings of this metabolic reprogramming have been described, its role in tumor progression is still under investigation. Importantly, the mitochondrion is a central actor in many of the metabolic processes that are altered in tumors. Yet, we have only begun to understand the dualities of mitochondrial function during cancer metastasis and therapy resistance. Paradoxically, mitochondrial metabolism can be both advantageous and detrimental to these processes, highlighting the need for a better understanding of the molecular and microenvironmental cues that define the role of this fascinating organelle. In this review article, we present an updated view on the different mitochondrial metabolic strategies adopted by cancer cells to overcome the many hurdles faced during tumor progression.

Extracellular vesicles are independent metabolic units with asparaginase activity.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. Although it has been shown that cells can traffic metabolic enzymes via EVs, much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Our metabolomics and functional analyses both revealed that EVs harbor L-asparaginase activity, catalyzed by the enzyme asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC EVs traffic Asrgl1. Our results demonstrate, for the first time, that NSC EVs function as independent metabolic units that are able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Tissue-specific and convergent metabolic transformation of cancer correlates with metastatic potential and patient survival.

Cancer cells undergo a multifaceted rewiring of cellular metabolism to support their biosynthetic needs. Although the major determinants of this metabolic transformation have been elucidated, their broad biological implications and clinical relevance are unclear. Here we systematically analyse the expression of metabolic genes across 20 different cancer types and investigate their impact on clinical outcome. We find that cancers undergo a tissue-specific metabolic rewiring, which converges towards a common metabolic landscape. Of note, downregulation of mitochondrial genes is associated with the worst clinical outcome across all cancer types and correlates with the expression of epithelial-to-mesenchymal transition gene signature, a feature of invasive and metastatic cancers. Consistently, suppression of mitochondrial genes is identified as a key metabolic signature of metastatic melanoma and renal cancer, and metastatic cell lines. This comprehensive analysis reveals unexpected facets of cancer metabolism, with important implications for cancer patients' stratification, prognosis and therapy.

Metabolic determinants of the immune modulatory function of neural stem cells.

BACKGROUND: Neural stem cells (NSCs) display tissue trophic and immune modulatory therapeutic activities after transplantation in central nervous system disorders. The intercellular interplay between stem cells and target immune cells is increased in NSCs exposed to inflammatory cues. Here, we hypothesize that inflammatory cytokine signalling leads to metabolic reprogramming of NSCs regulating some of their immune modulatory effects. METHODS: NSC lines were prepared from the subventricular zone (SVZ) of 7-12-week-old mice. Whole secretome-based screening and analysis of intracellular small metabolites was performed in NSCs exposed to cocktails of either Th1-like (IFN-γ, 500 U/ml; TNF-α, 200 U/ml; IL-1ß, 100 U/ml) or Th2-like (IL-4, IL-5 and IL-13; 10 ng/ml) inflammatory cytokines for 16 h in vitro. Isotopologues distribution of arginine and downstream metabolites was assessed by liquid chromatography/mass spectrometry in NSCs incubated with U-(13)C6 L-arginine in the presence or absence of Th1 or Th2 cocktails (Th1 NSCs or Th2 NSCs). The expression of arginase I and II was investigated in vitro in Th1 NSCs and Th2 NSCs and in vivo in the SVZ of mice with experimental autoimmune encephalomyelitis, as prototypical model of Th1 cell-driven brain inflammatory disease. The effects of the inflammatory cytokine signalling were studied in NSC-lymph node cells (LNC) co-cultures by flow cytometry-based analysis of cell proliferation following pan-arginase inhibition with N(ω)-hydroxy-nor-arginine (nor-NOHA). RESULTS: Cytokine-primed NSCs showed significantly higher anti-proliferative effect in co-cultures vs. control NSCs. Metabolomic analysis of intracellular metabolites revealed alteration of arginine metabolism and increased extracellular arginase I activity in cytokine-primed NSCs. Arginase inhibition by nor-NOHA partly rescued the anti-proliferative effects of cytokine-primed NSCs. CONCLUSIONS: Our work underlines the use of metabolic profiling as hypothesis-generating tools that helps unravelling how stem cell-mediated mechanisms of tissue restoration become affected by local inflammatory responses. Among different therapeutic candidates, we identify arginase signalling as novel metabolic determinant of the NSC-to-immune system communication.

Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition.

Mutations of the tricarboxylic acid cycle enzyme fumarate hydratase cause hereditary leiomyomatosis and renal cell cancer. Fumarate hydratase-deficient renal cancers are highly aggressive and metastasize even when small, leading to a very poor clinical outcome. Fumarate, a small molecule metabolite that accumulates in fumarate hydratase-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite. Fumarate has been shown to inhibit α-ketoglutarate-dependent dioxygenases that are involved in DNA and histone demethylation. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of fumarate hydratase and the subsequent accumulation of fumarate in mouse and human cells elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster mir-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of fumarate hydratase-proficient cells with cell-permeable fumarate. Loss of fumarate hydratase is associated with suppression of miR-200 and the EMT signature in renal cancer and is associated with poor clinical outcome. These results imply that loss of fumarate hydratase and fumarate accumulation contribute to the aggressive features of fumarate hydratase-deficient tumours.

Near-complete elimination of mutant mtDNA by iterative or dynamic dose-controlled treatment with mtZFNs.

Mitochondrial diseases are frequently associated with mutations in mitochondrial DNA (mtDNA). In most cases, mutant and wild-type mtDNAs coexist, resulting in heteroplasmy. The selective elimination of mutant mtDNA, and consequent enrichment of wild-type mtDNA, can rescue pathological phenotypes in heteroplasmic cells. Use of the mitochondrially targeted zinc finger-nuclease (mtZFN) results in degradation of mutant mtDNA through site-specific DNA cleavage. Here, we describe a substantial enhancement of our previous mtZFN-based approaches to targeting mtDNA, allowing near-complete directional shifts of mtDNA heteroplasmy, either by iterative treatment or through finely controlled expression of mtZFN, which limits off-target catalysis and undesired mtDNA copy number depletion. To demonstrate the utility of this improved approach, we generated an isogenic distribution of heteroplasmic cells with variable mtDNA mutant level from the same parental source without clonal selection. Analysis of these populations demonstrated an altered metabolic signature in cells harbouring decreased levels of mutant m.8993T>G mtDNA, associated with neuropathy, ataxia, and retinitis pigmentosa (NARP). We conclude that mtZFN-based approaches offer means for mtDNA heteroplasmy manipulation in basic research, and may provide a strategy for therapeutic intervention in selected mitochondrial diseases.