Genetic screening and metabolomics identify glial adenosine metabolism as a therapeutic target in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder and lacks disease-modifying therapies. We developed a Drosophila model for identifying novel glial-based therapeutic targets for PD. Human alpha-synuclein is expressed in neurons and individual genes are independently knocked down in glia. We performed a forward genetic screen, knocking down the entire Drosophila kinome in glia in alpha-synuclein expressing flies. Among the top hits were five genes (Ak1, Ak6, Adk1, Adk2, and awd) involved in adenosine metabolism. Knockdown of each gene improved locomotor dysfunction, rescued neurodegeneration, and increased brain adenosine levels. We determined that the mechanism of neuroprotection involves adenosine itself, as opposed to a downstream metabolite. We dove deeper into the mechanism for one gene, Ak1, finding rescue of dopaminergic neuron loss, alpha-synuclein aggregation, and bioenergetic dysfunction after glial Ak1 knockdown. We performed metabolomics in Drosophila and in human PD patients, allowing us to comprehensively characterize changes in purine metabolism and identify potential biomarkers of dysfunctional adenosine metabolism in people. These experiments support glial adenosine as a novel therapeutic target in PD.

Chromatin regulator SMARCAL1 modulates cellular lipid metabolism.

Biallelic mutations of the chromatin regulator SMARCAL1 cause Schimke Immunoosseous Dysplasia (SIOD), characterized by severe growth defects and premature mortality. Atherosclerosis and hyperlipidemia are common among SIOD patients, yet their onset and progression are poorly understood. Using an integrative approach involving proteomics, mouse models, and population genetics, we investigated SMARCAL1's role. We found that SmarcAL1 interacts with angiopoietin-like 3 (Angptl3), a key regulator of lipoprotein metabolism. In vitro and in vivo analyses demonstrate SmarcAL1's vital role in maintaining cellular lipid homeostasis. The observed translocation of SmarcAL1 to cytoplasmic peroxisomes suggests a potential regulatory role in lipid metabolism through gene expression. SmarcAL1 gene inactivation reduces the expression of key genes in cellular lipid catabolism. Population genetics investigations highlight significant associations between SMARCAL1 genetic variations and body mass index, along with lipid-related traits. This study underscores SMARCAL1's pivotal role in cellular lipid metabolism, likely contributing to the observed lipid phenotypes in SIOD patients.

Tryptophan metabolism determines outcome in tuberculous meningitis: a targeted metabolomic analysis.

Background: Cellular metabolism is critical for the host immune function against pathogens, and metabolomic analysis may help understand the characteristic immunopathology of tuberculosis. We performed targeted metabolomic analyses in a large cohort of patients with tuberculous meningitis (TBM), the most severe manifestation of tuberculosis, focusing on tryptophan metabolism. Methods: We studied 1069 Indonesian and Vietnamese adults with TBM (26.6% HIV-positive), 54 non-infectious controls, 50 with bacterial meningitis, and 60 with cryptococcal meningitis. Tryptophan and downstream metabolites were measured in cerebrospinal fluid (CSF) and plasma using targeted liquid chromatography-mass spectrometry. Individual metabolite levels were associated with survival, clinical parameters, CSF bacterial load and 92 CSF inflammatory proteins. Results: CSF tryptophan was associated with 60-day mortality from TBM (hazard ratio [HR] = 1.16, 95% confidence interval [CI] = 1.10-1.24, for each doubling in CSF tryptophan) both in HIV-negative and -positive patients. CSF tryptophan concentrations did not correlate with CSF bacterial load nor CSF inflammation but were negatively correlated with CSF interferon-gamma concentrations. Unlike tryptophan, CSF concentrations of an intercorrelating cluster of downstream kynurenine metabolites did not predict mortality. These CSF kynurenine metabolites did however correlate with CSF inflammation and markers of blood-CSF leakage, and plasma kynurenine predicted death (HR 1.54, 95% CI = 1.22-1.93). These findings were mostly specific for TBM, although high CSF tryptophan was also associated with mortality from cryptococcal meningitis. Conclusions: TBM patients with a high baseline CSF tryptophan or high systemic (plasma) kynurenine are at increased risk of death. These findings may reveal new targets for host-directed therapy. Funding: This study was supported by National Institutes of Health (R01AI145781) and the Wellcome Trust (110179/Z/15/Z and 206724/Z/17/Z).

The IDOze Study: The Link Between Sleep Disruption and Tryptophan-Kynurenine Pathway Activation in Women With Human Immunodeficiency Virus.

BACKGROUND: Poor sleep is associated with human immunodeficiency virus (HIV), particularly among women with HIV (WWH), although mechanisms are unclear. We explored cross-sectional associations between sleep disruption and tryptophan-kynurenine (T/K) pathway activation, measured by the kynurenine-to-tryptophan ratio (K:T). METHODS: HIV-uninfected women (HIV-) and WWH aged 35-70 years and on stable antiretroviral therapy were included. Sleep metrics were measured using wrist actigraphy. Plasma T/K pathway metabolites were measured using liquid chromatography-tandem mass spectrometry. Multivariate linear regression models examined relationships between K:T and actigraphy-based sleep metrics by HIV status. RESULTS: WWH (n = 153) and HIV- women (n = 151) were demographically similar. Among WWH, median CD4 was 751 cells/µL; 92% had undetectable HIV RNA. Compared to HIV- women, WWH had higher K:T (P < .001) and kynurenine (P = .01) levels but similar tryptophan levels (P = .25). Higher K:T was associated with more wake bouts (P = .001), more time awake after sleep onset (P = .01), and lower sleep efficiency (P = .03) in WWH only. CONCLUSIONS: HIV infection was associated with T/K pathway activation; this activation was associated with poorer sleep efficiency and more fragmented sleep. While longitudinal studies are needed to elucidate the directionality of these associations, these findings may help identify treatments to reduce sleep disruption in WWH by targeting residual inflammation and T/K pathway activation.

Associations With Extubation Failure and Predictive Value of Risk Analytics Algorithms With Extubation Readiness Tests Following Congenital Cardiac Surgery.

OBJECTIVES: Extubation failure is associated with morbidity and mortality in children following cardiac surgery. Current extubation readiness tests (ERT) do not consider the nonrespiratory support provided by mechanical ventilation (MV) for children with congenital heart disease. We aimed to identify factors associated with extubation failure in children following cardiac surgery and assess the performance of two risk analytics algorithms for patients undergoing an ERT. DESIGN: Retrospective cohort study. SETTING: CICU at a tertiary-care children's hospital. PATIENTS: Children receiving MV greater than 48 hours following cardiac surgery between January 1, 2017, and December 31, 2019. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Six hundred fifty encounters were analyzed with 49 occurrences (8%) of reintubation. Extubation failure occurred most frequently within 6 hours of extubation. On multivariable analysis, younger age (per each 3-mo decrease: odds ratio [OR], 1.06; 95% CI, 1.001-1.12), male sex (OR, 2.02; 95% CI, 1.03-3.97), Society of Thoracic Surgery-European Association for Cardiothoracic Surgery category 5 procedure (p equals to 0.005), and preoperative respiratory support (OR, 2.08; 95% CI, 1.09-3.95) were independently associated with unplanned reintubation. Our institutional ERT had low sensitivity to identify patients at risk for reintubation (23.8%; 95% CI, 9.7-47.6%). The addition of the inadequate delivery of oxygen (IDO2) index to the ERT increased the sensitivity by 19.0% (95% CI, -2.5 to 40.7%; p = 0.05), but the sensitivity remained low and the accuracy of the test dropped by 8.9% (95% CI, 4.7-13.1%; p < 0.01). CONCLUSIONS: Preoperative respiratory support, younger age, and more complex operations are associated with postoperative extubation failure. IDO2 and IVCO2 provide unique cardiorespiratory monitoring parameters during ERTs but require further investigation before being used in clinical evaluation for extubation failure.

Portal Venous Metabolite Profiling After RYGB in Male Rats Highlights Changes in Gut-Liver Axis.

After Roux-en-Y gastric bypass (RYGB) surgery, the intestine undergoes structural and metabolic reprogramming and appears to enhance use of energetic fuels including glucose and amino acids (AAs), changes that may be related to the surgery's remarkable metabolic effects. Consistently, RYGB alters serum levels of AAs and other metabolites, perhaps reflecting mechanisms for metabolic improvement. To home in on the intestinal contribution, we performed metabolomic profiling in portal venous (PV) blood from lean, Long Evans rats after RYGB vs sham surgery. We found that one-carbon metabolism (OCM), nitrogen metabolism, and arginine and proline metabolism were significantly enriched in PV blood. Nitrogen, OCM, and sphingolipid metabolism as well as ubiquinone biosynthesis were also overrepresented among metabolites uniquely affected in PV vs peripheral blood in RYGB-operated but not sham-operated animals. Peripheral blood demonstrated changes in AA metabolism, OCM, sphingolipid metabolism, and glycerophospholipid metabolism. Despite enrichment for many of the same pathways, the overall metabolite fingerprint of the 2 compartments did not correlate, highlighting a unique role for PV metabolomic profiling as a window into gut metabolism. AA metabolism and OCM were enriched in peripheral blood both from humans and lean rats after RYGB, demonstrating that these conserved pathways might represent mechanisms for clinical improvement elicited by the surgery in patients. Together, our data provide novel insight into RYGB's effects on the gut-liver axis and highlight a role for OCM as a key metabolic pathway affected by RYGB.

A Prospective Analysis of Circulating Plasma Metabolites Associated with Ovarian Cancer Risk.

Ovarian cancer has few known risk factors, hampering identification of high-risk women. We assessed the association of prediagnostic plasma metabolites (N = 420) with risk of epithelial ovarian cancer, including both borderline and invasive tumors. A total of 252 cases and 252 matched controls from the Nurses' Health Studies were included. Multivariable logistic regression was used to estimate ORs and 95% confidence intervals (CI), comparing the 90th-10th percentile in metabolite levels, using the permutation-based Westfall and Young approach to account for testing multiple correlated hypotheses. Weighted gene coexpression network analysis (WGCNA; n = 10 metabolite modules) and metabolite set enrichment analysis (n = 23 metabolite classes) were also evaluated. An increase in pseudouridine levels from the 10th to the 90th percentile was associated with a 2.5-fold increased risk of overall ovarian cancer (OR = 2.56; 95% CI, 1.48-4.45; P = 0.001/adjusted P = 0.15); a similar risk estimate was observed for serous/poorly differentiated tumors (n = 176 cases; comparable OR = 2.38; 95% CI, 1.33-4.32; P = 0.004/adjusted P = 0.55). For nonserous tumors (n = 34 cases), pseudouridine and C36:2 phosphatidylcholine plasmalogen had the strongest statistical associations (OR = 9.84; 95% CI, 2.89-37.82; P < 0.001/adjusted P = 0.07; and OR = 0.11; 95% CI, 0.03-0.35; P < 0.001/adjusted P = 0.06, respectively). Five WGCNA modules and 9 classes were associated with risk overall at FDR ≤ 0.20. Triacylglycerols (TAG) showed heterogeneity by tumor aggressiveness (case-only heterogeneity P < 0.0001). The TAG association with risk overall and serous tumors differed by acyl carbon content and saturation. In summary, this study suggests that pseudouridine may be a novel risk factor for ovarian cancer and that TAGs may also be important, particularly for rapidly fatal tumors, with associations differing by structural features. SIGNIFICANCE: Pseudouridine represents a potential novel risk factor for ovarian cancer and triglycerides may be important particularly in rapidly fatal ovarian tumors.

Metabolomic adaptations and correlates of survival to immune checkpoint blockade.

Despite remarkable success of immune checkpoint inhibitors, the majority of cancer patients have yet to receive durable benefits. Here, in order to investigate the metabolic alterations in response to immune checkpoint blockade, we comprehensively profile serum metabolites in advanced melanoma and renal cell carcinoma patients treated with nivolumab, an antibody against programmed cell death protein 1 (PD1). We identify serum kynurenine/tryptophan ratio increases as an adaptive resistance mechanism associated with worse overall survival. This advocates for patient stratification and metabolic monitoring in immunotherapy clinical trials including those combining PD1 blockade with indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase   (IDO/TDO) inhibitors.

Gut microbiome structure and metabolic activity in inflammatory bowel disease.

The inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are multifactorial chronic conditions of the gastrointestinal tract. While IBD has been associated with dramatic changes in the gut microbiota, changes in the gut metabolome-the molecular interface between host and microbiota-are less well understood. To address this gap, we performed untargeted metabolomic and shotgun metagenomic profiling of cross-sectional stool samples from discovery (n = 155) and validation (n = 65) cohorts of CD, UC and non-IBD control patients. Metabolomic and metagenomic profiles were broadly correlated with faecal calprotectin levels (a measure of gut inflammation). Across >8,000 measured metabolite features, we identified chemicals and chemical classes that were differentially abundant in IBD, including enrichments for sphingolipids and bile acids, and depletions for triacylglycerols and tetrapyrroles. While > 50% of differentially abundant metabolite features were uncharacterized, many could be assigned putative roles through metabolomic 'guilt by association' (covariation with known metabolites). Differentially abundant species and functions from the metagenomic profiles reflected adaptation to oxidative stress in the IBD gut, and were individually consistent with previous findings. Integrating these data, however, we identified 122 robust associations between differentially abundant species and well-characterized differentially abundant metabolites, indicating possible mechanistic relationships that are perturbed in IBD. Finally, we found that metabolome- and metagenome-based classifiers of IBD status were highly accurate and, like the vast majority of individual trends, generalized well to the independent validation cohort. Our findings thus provide an improved understanding of perturbations of the microbiome-metabolome interface in IBD, including identification of many potential diagnostic and therapeutic targets.

Aneuploid Cell Survival Relies upon Sphingolipid Homeostasis.

Aneuploidy, a hallmark of cancer cells, poses an appealing opportunity for cancer treatment and prevention strategies. Using a cell-based screen to identify small molecules that could selectively kill aneuploid cells, we identified the compound N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl]-decanamide monohydrochloride (DL-PDMP), an antagonist of UDP-glucose ceramide glucosyltransferase. DL-PDMP selectively inhibited proliferation of aneuploid primary mouse embryonic fibroblasts and aneuploid colorectal cancer cells. Its selective cytotoxic effects were based on further accentuating the elevated levels of ceramide, which characterize aneuploid cells, leading to increased apoptosis. We observed that DL-PDMP could also enhance the cytotoxic effects of paclitaxel, a standard-of-care chemotherapeutic agent that causes aneuploidy, in human colon cancer and mouse lymphoma cells. Our results offer pharmacologic evidence that the aneuploid state in cancer cells can be targeted selectively for therapeutic purposes, or for reducing the toxicity of taxane-based drug regimens. Cancer Res; 77(19); 5272-86. ©2017 AACR.

Distinct metabolomic signatures are associated with longevity in humans.

Alterations in metabolism influence lifespan in experimental models, but data in humans are lacking. Here we use liquid chromatography/mass spectrometry to quantify 217 plasma metabolites and examine their relation to longevity in a large cohort of men and women followed for up to 20 years. We find that, higher concentrations of the citric acid cycle intermediate, isocitrate, and the bile acid, taurocholate, are associated with lower odds of longevity, defined as attaining 80 years of age. Higher concentrations of isocitrate, but not taurocholate, are also associated with worse cardiovascular health at baseline, as well as risk of future cardiovascular disease and death. None of the metabolites identified are associated with cancer risk. Our findings suggest that some, but not all, metabolic pathways related to human longevity are linked to the risk of common causes of death.

Estimating glutathione synthesis with deuterated water: a model for peptide biosynthesis.

Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of (2)H from orally supplied (2)H(2)O into stable C-H bonds on the tripeptide. The incorporation of (2)H(2)O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with (2)H(2)O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N=6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with (2)H(2)O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of (2)H(2)O and a statistical basis for estimating N.