A glycolytic metabolite bypasses "two-hit" tumor suppression by BRCA2.

Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.

Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages.

Activated macrophages undergo metabolic reprogramming, which drives their pro-inflammatory phenotype, but the mechanistic basis for this remains obscure. Here, we demonstrate that upon lipopolysaccharide (LPS) stimulation, macrophages shift from producing ATP by oxidative phosphorylation to glycolysis while also increasing succinate levels. We show that increased mitochondrial oxidation of succinate via succinate dehydrogenase (SDH) and an elevation of mitochondrial membrane potential combine to drive mitochondrial reactive oxygen species (ROS) production. RNA sequencing reveals that this combination induces a pro-inflammatory gene expression profile, while an inhibitor of succinate oxidation, dimethyl malonate (DMM), promotes an anti-inflammatory outcome. Blocking ROS production with rotenone by uncoupling mitochondria or by expressing the alternative oxidase (AOX) inhibits this inflammatory phenotype, with AOX protecting mice from LPS lethality. The metabolic alterations that occur upon activation of macrophages therefore repurpose mitochondria from ATP synthesis to ROS production in order to promote a pro-inflammatory state.

Fumarate induces vesicular release of mtDNA to drive innate immunity.

Mutations in fumarate hydratase (FH) cause hereditary leiomyomatosis and renal cell carcinoma1. Loss of FH in the kidney elicits several oncogenic signalling cascades through the accumulation of the oncometabolite fumarate2. However, although the long-term consequences of FH loss have been described, the acute response has not so far been investigated. Here we generated an inducible mouse model to study the chronology of FH loss in the kidney. We show that loss of FH leads to early alterations of mitochondrial morphology and the release of mitochondrial DNA (mtDNA) into the cytosol, where it triggers the activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING)-TANK-binding kinase 1 (TBK1) pathway and stimulates an inflammatory response that is also partially dependent on retinoic-acid-inducible gene I (RIG-I). Mechanistically, we show that this phenotype is mediated by fumarate and occurs selectively through mitochondrial-derived vesicles in a manner that depends on sorting nexin 9 (SNX9). These results reveal that increased levels of intracellular fumarate induce a remodelling of the mitochondrial network and the generation of mitochondrial-derived vesicles, which allows the release of mtDNAin the cytosol and subsequent activation of the innate immune response.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

Mitochondria-Endoplasmic Reticulum Contact Sites Function as Immunometabolic Hubs that Orchestrate the Rapid Recall Response of Memory CD8+ T Cells.

Glycolysis is linked to the rapid response of memory CD8+ T cells, but the molecular and subcellular structural elements enabling enhanced glucose metabolism in nascent activated memory CD8+ T cells are unknown. We found that rapid activation of protein kinase B (PKB or AKT) by mammalian target of rapamycin complex 2 (mTORC2) led to inhibition of glycogen synthase kinase 3ß (GSK3ß) at mitochondria-endoplasmic reticulum (ER) junctions. This enabled recruitment of hexokinase I (HK-I) to the voltage-dependent anion channel (VDAC) on mitochondria. Binding of HK-I to VDAC promoted respiration by facilitating metabolite flux into mitochondria. Glucose tracing pinpointed pyruvate oxidation in mitochondria, which was the metabolic requirement for rapid generation of interferon-γ (IFN-γ) in memory T cells. Subcellular organization of mTORC2-AKT-GSK3ß at mitochondria-ER contact sites, promoting HK-I recruitment to VDAC, thus underpins the metabolic reprogramming needed for memory CD8+ T cells to rapidly acquire effector function.

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.

Signaling metabolite L-2-hydroxyglutarate activates the transcription factor HIF-1α in lipopolysaccharide-activated macrophages.

Activated macrophages undergo metabolic reprogramming, which not only supports their energetic demands but also allows for the production of specific metabolites that function as signaling molecules. Several Krebs cycles, or Krebs-cycle-derived metabolites, including succinate, α-ketoglutarate, and itaconate, have recently been shown to modulate macrophage function. The accumulation of 2-hydroxyglutarate (2HG) has also been well documented in transformed cells and more recently shown to play a role in T cell and dendritic cell function. Here we have found that the abundance of both enantiomers of 2HG is increased in LPS-activated macrophages. We show that L-2HG, but not D-2HG, can promote the expression of the proinflammatory cytokine IL-1ß and the adoption of an inflammatory, highly glycolytic metabolic state. These changes are likely mediated through activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) by L-2HG, a known inhibitor of the HIF prolyl hydroxylases. Expression of the enzyme responsible for L-2HG degradation, L-2HG dehydrogenase (L-2HGDH), was also found to be decreased in LPS-stimulated macrophages and may therefore also contribute to L-2HG accumulation. Finally, overexpression of L-2HGDH in HEK293 TLR4/MD2/CD14 cells inhibited HIF-1α activation by LPS, while knockdown of L-2HGDH in macrophages boosted the induction of HIF-1α-dependent genes, as well as increasing LPS-induced HIF-1α activity. Taken together, this study therefore identifies L-2HG as a metabolite that can regulate HIF-1α in macrophages.

Oral famotidine versus placebo in non-hospitalised patients with COVID-19: a randomised, double-blind, data-intense, phase 2 clinical trial.

OBJECTIVE: We assessed whether famotidine improved inflammation and symptomatic recovery in outpatients with mild to moderate COVID-19. DESIGN: Randomised, double-blind, placebo-controlled, fully remote, phase 2 clinical trial (NCT04724720) enrolling symptomatic unvaccinated adult outpatients with confirmed COVID-19 between January 2021 and April 2021 from two US centres. Patients self-administered 80 mg famotidine (n=28) or placebo (n=27) orally three times a day for 14 consecutive days. Endpoints were time to (primary) or rate of (secondary) symptom resolution, and resolution of inflammation (exploratory). RESULTS: Of 55 patients in the intention-to-treat group (median age 35 years (IQR: 20); 35 women (64%); 18 African American (33%); 14 Hispanic (26%)), 52 (95%) completed the trial, submitting 1358 electronic symptom surveys. Time to symptom resolution was not statistically improved (p=0.4). Rate of symptom resolution was improved for patients taking famotidine (p<0.0001). Estimated 50% reduction of overall baseline symptom scores were achieved at 8.2 days (95% CI: 7 to 9.8 days) for famotidine and 11.4 days (95% CI: 10.3 to 12.6 days) for placebo treated patients. Differences were independent of patient sex, race or ethnicity. Five self-limiting adverse events occurred (famotidine, n=2 (40%); placebo, n=3 (60%)). On day 7, fewer patients on famotidine had detectable interferon alpha plasma levels (p=0.04). Plasma immunoglobulin type G levels to SARS-CoV-2 nucleocapsid core protein were similar between both arms. CONCLUSIONS: Famotidine was safe and well tolerated in outpatients with mild to moderate COVID-19. Famotidine led to earlier resolution of symptoms and inflammation without reducing anti-SARS-CoV-2 immunity. Additional randomised trials are required.

BCAT1 affects mitochondrial metabolism independently of leucine transamination in activated human macrophages.

In response to environmental stimuli, macrophages change their nutrient consumption and undergo an early metabolic adaptation that progressively shapes their polarization state. During the transient, early phase of pro-inflammatory macrophage activation, an increase in tricarboxylic acid (TCA) cycle activity has been reported, but the relative contribution of branched-chain amino acid (BCAA) leucine remains to be determined. Here, we show that glucose but not glutamine is a major contributor of the increase in TCA cycle metabolites during early macrophage activation in humans. We then show that, although uptake of BCAAs is not altered, their transamination by BCAT1 is increased following 8 h lipopolysaccharide (LPS) stimulation. Of note, leucine is not metabolized to integrate into the TCA cycle in basal or stimulated human macrophages. Surprisingly, the pharmacological inhibition of BCAT1 reduced glucose-derived itaconate, α-ketoglutarate and 2-hydroxyglutarate levels without affecting succinate and citrate levels, indicating a partial inhibition of the TCA cycle. This indirect effect is associated with NRF2 (also known as NFE2L2) activation and anti-oxidant responses. These results suggest a moonlighting role of BCAT1 through redox-mediated control of mitochondrial function during early macrophage activation.

Causal integration of multi-omics data with prior knowledge to generate mechanistic hypotheses.

Multi-omics datasets can provide molecular insights beyond the sum of individual omics. Various tools have been recently developed to integrate such datasets, but there are limited strategies to systematically extract mechanistic hypotheses from them. Here, we present COSMOS (Causal Oriented Search of Multi-Omics Space), a method that integrates phosphoproteomics, transcriptomics, and metabolomics datasets. COSMOS combines extensive prior knowledge of signaling, metabolic, and gene regulatory networks with computational methods to estimate activities of transcription factors and kinases as well as network-level causal reasoning. COSMOS provides mechanistic hypotheses for experimental observations across multi-omics datasets. We applied COSMOS to a dataset comprising transcriptomics, phosphoproteomics, and metabolomics data from healthy and cancerous tissue from eleven clear cell renal cell carcinoma (ccRCC) patients. COSMOS was able to capture relevant crosstalks within and between multiple omics layers, such as known ccRCC drug targets. We expect that our freely available method will be broadly useful to extract mechanistic insights from multi-omics studies.

Glutaminolysis is a metabolic dependency in FLT3ITD acute myeloid leukemia unmasked by FLT3 tyrosine kinase inhibition.

FLT3 internal tandem duplication (FLT3ITD) mutations are common in acute myeloid leukemia (AML) associated with poor patient prognosis. Although new-generation FLT3 tyrosine kinase inhibitors (TKI) have shown promising results, the outcome of FLT3ITD AML patients remains poor and demands the identification of novel, specific, and validated therapeutic targets for this highly aggressive AML subtype. Utilizing an unbiased genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screen, we identify GLS, the first enzyme in glutamine metabolism, as synthetically lethal with FLT3-TKI treatment. Using complementary metabolomic and gene-expression analysis, we demonstrate that glutamine metabolism, through its ability to support both mitochondrial function and cellular redox metabolism, becomes a metabolic dependency of FLT3ITD AML, specifically unmasked by FLT3-TKI treatment. We extend these findings to AML subtypes driven by other tyrosine kinase (TK) activating mutations and validate the role of GLS as a clinically actionable therapeutic target in both primary AML and in vivo models. Our work highlights the role of metabolic adaptations as a resistance mechanism to several TKI and suggests glutaminolysis as a therapeutically targetable vulnerability when combined with specific TKI in FLT3ITD and other TK activating mutation-driven leukemias.

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

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.

Bilingualism in Primary Progressive Aphasia: A Retrospective Study on Clinical and Language Characteristics.

BACKGROUND: Primary progressive aphasia (PPA) is a neurodegenerative disorder characterized by progressive deterioration of language. Being rare, reports of PPA in multilingual individuals are scarce, despite more than half of the world population being multilingual. METHODS: We describe clinical characteristics of 33 bilingual patients with PPA, including symptom presentation and language deficits pattern in their first (L1) and second language (L2), through a systematic literature review and new cases retrospectively identified in 5 countries. RESULTS: In total, 14 patients presented with nonfluent/agrammatic variant, 6 with semantic variant, and 13 with logopenic variant, with a median symptom onset of 2 years. Word-finding difficulties was the first symptom in 65% of all cases, initially noticed in L2, and not always the dominant language. Our group had 22 different languages as L1, and 9 as L2. At the whole-group level there was a tendency for parallel impairment in both languages, in line with the shared bilingual neural substrate hypothesis, but each PPA variant showed some heterogeneity. DISCUSSION: Each PPA variant showed heterogeneity, showing the need for comprehensive language and cognitive assessment across languages, as well as further clarification on the role of language mediators.

Ischemic preconditioning protects against cardiac ischemia reperfusion injury without affecting succinate accumulation or oxidation.

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted and then restored, and underlies many disorders, notably myocardial infarction and stroke. While reperfusion of ischemic tissue is essential for survival, it also initiates cell death through generation of mitochondrial reactive oxygen species (ROS). Recent work has revealed a novel pathway underlying ROS production at reperfusion in vivo in which the accumulation of succinate during ischemia and its subsequent rapid oxidation at reperfusion drives ROS production at complex I by reverse electron transport (RET). Pharmacologically inhibiting ischemic succinate accumulation, or slowing succinate metabolism at reperfusion, have been shown to be cardioprotective against IR injury. Here, we determined whether ischemic preconditioning (IPC) contributes to cardioprotection by altering kinetics of succinate accumulation and oxidation during IR. Mice were subjected to a 30-minute occlusion of the left anterior descending coronary artery followed by reperfusion, with or without a protective IPC protocol prior to sustained ischemia. We found that IPC had no effect on ischemic succinate accumulation with both control and IPC mice having profound increases in succinate compared to normoxia. Furthermore, after only 1-minute reperfusion succinate was rapidly metabolised returning to near pre-ischemic levels in both groups. We conclude that IPC does not affect ischemic succinate accumulation, or its oxidation at reperfusion.

Post-translational regulation of metabolism in fumarate hydratase deficient cancer cells.

Deregulated signal transduction and energy metabolism are hallmarks of cancer and both play a fundamental role in tumorigenesis. While it is increasingly recognised that signalling and metabolism are highly interconnected, the underpinning mechanisms of their co-regulation are still largely unknown. Here we designed and acquired proteomics, phosphoproteomics, and metabolomics experiments in fumarate hydratase (FH) deficient cells and developed a computational modelling approach to identify putative regulatory phosphorylation-sites of metabolic enzymes. We identified previously reported functionally relevant phosphosites and potentially novel regulatory residues in enzymes of the central carbon metabolism. In particular, we showed that pyruvate dehydrogenase (PDHA1) enzymatic activity is inhibited by increased phosphorylation in FH-deficient cells, restricting carbon entry from glucose to the tricarboxylic acid cycle. Moreover, we confirmed PDHA1 phosphorylation in human FH-deficient tumours. Our work provides a novel approach to investigate how post-translational modifications of enzymes regulate metabolism and could have important implications for understanding the metabolic transformation of FH-deficient cancers with potential clinical applications.

Clinical predictors of individual cognitive fluctuations in patients undergoing hemodialysis.

BACKGROUND: Cognitive impairment in hemodialysis (HD) patients is frequent and mediated by several factors. It is unclear which patients are more susceptible to cognitive variations around the dialysis cycle and which clinical factors may play a mediator role. We aimed to answer these issues by investigating intraindividual changes within the dialysis cycle. STUDY DESIGN: Cross-sectional observational study with repeated measures. SETTING & PARTICIPANTS: 47 HD patients and 40 controls without kidney disease, both without history of neurologic disease. PREDICTORS: Dialysis vintage, disease duration, vascular risk factors, comorbidity index score, intradialytic weight change, frequency of hypotensive episodes, and biochemical levels (hemoglobin, leukocytes, urea, creatinine, sodium, and potassium). Covariates included demographics (age, education, and sex). OUTCOMES & MEASUREMENTS: Significant individual deterioration in attention and executive functions (phasic and intrinsic alertness, Stroop test, and Trail Making Test) after dialysis, as measured by a regression-based reliable change method. Regression models were used to identify clinical predictors of individual cognitive decline after dialysis. RESULTS: After dialysis, patients primarily showed prolonged reaction times and psychomotor slowing. However, individual-based analyses revealed that fluctuations in attention and executive functions were present in only a minority of patients. Significant individual fluctuations on particular attention and executive tasks were associated moderately with intradialytic hypotensive episodes, as well as with psychoactive medication, and were predicted weakly by blood leukocyte count, sodium level, dialysis vintage, and volume. LIMITATIONS: Small sample size; patient group younger and healthier than the overall HD population, limiting generalizability. CONCLUSIONS: Only a minority of patients exhibit significant individual cognitive fluctuations, predominantly showing deterioration after dialysis in attention and executive functions. Susceptibility to such fluctuations was predicted in part by both HD-dependent and -independent factors.

Genotype-specific patterns of atrophy progression are more sensitive than clinical decline in SCA1, SCA3 and SCA6.

Spinocerebellar ataxias are dominantly inherited disorders that are associated with progressive brain degeneration, mainly affecting the cerebellum and brainstem. As part of the multicentre European integrated project on spinocerebellar ataxias study, 37 patients with spinocerebellar ataxia-1, 19 with spinocerebellar ataxia-3 and seven with spinocerebellar ataxia-6 were clinically examined and underwent magnetic resonance imaging at baseline and after a 2-year follow-up. All patients were compared with age-matched and gender-matched healthy control subjects. Magnetic resonance imaging analysis included three-dimensional volumetry and observer-independent longitudinal voxel-based morphometry. Volumetry revealed loss of brainstem, cerebellar and basal ganglia volume in all genotypes. Most sensitive to change was the pontine volume in spinocerebellar ataxia-1, striatal volume in spinocerebellar ataxia-3 and caudate volume in spinocerebellar ataxia-6. Sensitivity to change, as measured by standard response mean, of the respective MRI measures was greater than that of the most sensitive clinical measure, the Scale for the Assessment and Rating of Ataxia. Longitudinal voxel-based morphometry revealed greatest grey matter loss in the cerebellum and brainstem in spinocerebellar ataxia-1, in the putamen and pallidum in spinocerebellar ataxia-3 and in the cerebellum, thalamus, putamen and pallidum in spinocerebellar ataxia-6. There was a mild correlation between CAG repeat length and volume loss of the bilateral cerebellum and the pons in spinocerebellar ataxia-1. Quantitative volumetry and voxel-based morphometry imaging demonstrated genotype-specific patterns of atrophy progression in spinocerebellar ataxias-1, 3 and 6, and they showed a high sensitivity to detect change that was superior to clinical scales. These structural magnetic resonance imaging findings have the potential to serve as surrogate markers, which might help to delineate quantifiable endpoints and non-invasive methods for rapid and reliable data acquisition, encouraging their use in clinical trials.

Characteristic functional connectome related to Post-COVID-19 syndrome.

Post-COVID-19 syndrome is a serious complication following SARS-CoV-2 infection, characterized primarily by fatigue and cognitive complaints. Although first metabolic and structural imaging alterations in Post-COVID-19 syndrome have been identified, their functional consequences remain unknown. Thus, we explored the impact of Post-COVID-19 syndrome on the functional connectome of the brain providing a deeper understanding of pathophysiological mechanisms. In a cross-sectional observational study, resting-state functional magnetic resonance imaging data of 66 patients with Post-COVID-19 syndrome after mild infection (mean age 42.3 years, 57 female) and 57 healthy controls (mean age 42.1 years, 38 female) with a mean time of seven months after acute COVID-19 were analysed using a graph theoretical approach. Network features were quantified using measures including mean distance, nodal degree, betweenness and Katz centrality, and compared between both groups. Graph measures were correlated with clinical measures quantifying fatigue, cognitive function, affective symptoms and sleep disturbances. Alterations were mainly found in the brainstem, olfactory cortex, cingulate cortex, thalamus and cerebellum on average seven months after SARS-CoV-2 infection. Additionally, strong correlations between fatigue severity, cognitive functioning and daytime sleepiness from clinical scales and graph measures were observed. Our study confirms functional relevance of brain imaging changes in Post-COVID-19 syndrome as mediating factors for persistent symptoms and improves our pathophysiological understanding.