A Data Mining Metabolomics Exploration of Glaucoma.

Glaucoma is an age related disease characterized by the progressive loss of retinal ganglion cells, which are the neurons that transduce the visual information from the retina to the brain. It is the leading cause of irreversible blindness worldwide. To gain further insights into primary open-angle glaucoma (POAG) pathophysiology, we performed a non-targeted metabolomics analysis on the plasma from POAG patients (n = 34) and age- and sex-matched controls (n = 30). We investigated the differential signature of POAG plasma compared to controls, using liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS). A data mining strategy, combining a filtering method with threshold criterion, a wrapper method with iterative selection, and an embedded method with penalization constraint, was used. These strategies are most often used separately in metabolomics studies, with each of them having their own limitations. We opted for a synergistic approach as a mean to unravel the most relevant metabolomics signature. We identified a set of nine metabolites, namely: nicotinamide, hypoxanthine, xanthine, and 1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline with decreased concentrations and N-acetyl-L-Leucine, arginine, RAC-glycerol 1-myristate, 1-oleoyl-RAC-glycerol, cystathionine with increased concentrations in POAG; the modification of nicotinamide, N-acetyl-L-Leucine, and arginine concentrations being the most discriminant. Our findings open up therapeutic perspectives for the diagnosis and treatment of POAG.

Tryptophane-kynurenine pathway in the remote ischemic conditioning mechanism.

The actual protective mechanisms underlying cardioprotection with remote ischemic conditioning (RIC) remain unclear. Recent data suggest that RIC induces kynurenine (KYN) and kynurenic acid synthesis, two metabolites derived from tryptophan (TRP), yet a causal relation between TRP pathway and RIC remains to be established. We sought to study the impact of RIC on the levels of TRP and its main metabolites within tissues, and to assess whether blocking kynurenine (KYN) synthesis from TRP would inhibit RIC-induced cardioprotection. In rats exposed to 40-min coronary occlusion and 2-h reperfusion, infarct size was significantly smaller in RIC-treated animals (35.7 ± 3.0% vs. 46.5 ± 2.2%, p = 0.01). This protection was lost in rats that received 1-methyl-tryptophan (1-MT) pretreatment, an inhibitor of KYN synthesis from TRP (infarct size = 46.2 ± 5.0%). Levels of TRP and nine compounds spanning its metabolism through the serotonin and KYN pathways were measured by reversed-phase liquid chromatography-tandem mass spectrometry in the liver, heart, and limb skeletal muscle, either exposed or not to RIC. In the liver, RIC induced a significant increase in xanthurenic acid, nicotinic acid, and TRP. Likewise, RIC increased NAD-dependent deacetylase sirtuin activity in the liver. Pretreatment with 1-MT suppressed the RIC-induced increases in NAD-dependent deacetylase sirtuin activity. Altogether, these findings indicate that RIC mechanism is dependent on TRP-KYN pathway activation.

Lactic Acidosis Together with GM-CSF and M-CSF Induces Human Macrophages toward an Inflammatory Protumor Phenotype.

In established tumors, tumor-associated macrophages (TAM) orchestrate nonresolving cancer-related inflammation and produce mediators favoring tumor growth, metastasis, and angiogenesis. However, the factors conferring inflammatory and protumor properties on human macrophages remain largely unknown. Most solid tumors have high lactate content. We therefore analyzed the impact of lactate on human monocyte differentiation. We report that prolonged lactic acidosis induces the differentiation of monocytes into macrophages with a phenotype including protumor and inflammatory characteristics. These cells produce tumor growth factors, inflammatory cytokines, and chemokines as well as low amounts of IL10. These effects of lactate require its metabolism and are associated with hypoxia-inducible factor-1α stabilization. The expression of some lactate-induced genes is dependent on autocrine M-CSF consumption. Finally, TAMs with protumor and inflammatory characteristics (VEGFhigh CXCL8+ IL1ß+) are found in solid ovarian tumors. These results show that tumor-derived lactate links the protumor features of TAMs with their inflammatory properties. Treatments that reduce tumor glycolysis or tumor-associated acidosis may help combat cancer.

Nicotinamide Deficiency in Primary Open-Angle Glaucoma.

Purpose: To investigate the plasma concentration of nicotinamide in primary open-angle glaucoma (POAG). Methods: Plasma of 34 POAG individuals was compared to that of 30 age- and sex-matched controls using a semiquantitative method based on liquid chromatography coupled to high-resolution mass spectrometry. Subsequently, an independent quantitative method, based on liquid chromatography coupled to mass spectrometry, was used to assess nicotinamide concentration in the plasma from the same initial cohort and from a replicative cohort of 20 POAG individuals and 15 controls. Results: Using the semiquantitative method, the plasma nicotinamide concentration was significantly lower in the initial cohort of POAG individuals compared to controls and further confirmed in the same cohort, using the targeted quantitative method, with mean concentrations of 0.14 µM (median: 0.12 µM; range, 0.06-0.28 µM) in the POAG group (-30%; P = 0.022) and 0.19 µM (median: 0.18 µM; range, 0.08-0.47 µM) in the control group. The quantitative dosage also disclosed a significantly lower plasma nicotinamide concentration (-33%; P = 0.011) in the replicative cohort with mean concentrations of 0.14 µM (median: 0.14 µM; range, 0.09-0.25 µM) in the POAG group, and 0.19 µM (median: 0.21 µM; range, 0.09-0.26 µM) in the control group. Conclusions: Glaucoma is associated with lower plasmatic nicotinamide levels, compared to controls, suggesting that nicotinamide supplementation might become a future therapeutic strategy. Further studies are needed, in larger cohorts, to confirm these preliminary findings.

The Metabolomic Bioenergetic Signature of Opa1-Disrupted Mouse Embryonic Fibroblasts Highlights Aspartate Deficiency.

OPA1 (Optic Atrophy 1) is a multi-isoform dynamin GTPase involved in the regulation of mitochondrial fusion and organization of the cristae structure of the mitochondrial inner membrane. Pathogenic OPA1 variants lead to a large spectrum of disorders associated with visual impairment due to optic nerve neuropathy. The aim of this study was to investigate the metabolomic consequences of complete OPA1 disruption in Opa1-/- mouse embryonic fibroblasts (MEFs) compared to their Opa1+/+ counterparts. Our non-targeted metabolomics approach revealed significant modifications of the concentration of several mitochondrial substrates, i.e. a decrease of aspartate, glutamate and α-ketoglutaric acid, and an increase of asparagine, glutamine and adenosine-5'-monophosphate, all related to aspartate metabolism. The signature further highlighted the altered metabolism of nucleotides and NAD together with deficient mitochondrial bioenergetics, reflected by the decrease of creatine/creatine phosphate and pantothenic acid, and the increase in pyruvate and glutathione. Interestingly, we recently reported significant variations of five of these molecules, including aspartate and glutamate, in the plasma of individuals carrying pathogenic OPA1 variants. Our findings show that the disruption of OPA1 leads to a remodelling of bioenergetic pathways with the central role being played by aspartate and related metabolites.

A Plasma Metabolomic Signature Involving Purine Metabolism in Human Optic Atrophy 1 (OPA1)-Related Disorders.

Purpose: Dominant optic atrophy (DOA; MIM [Mendelian Inheritance in Man] 165500), resulting in retinal ganglion cell degeneration, is mainly caused by mutations in the optic atrophy 1 (OPA1) gene, which encodes a dynamin guanosine triphosphate (GTP)ase involved in mitochondrial membrane processing. This work aimed at determining whether plasma from OPA1 pathogenic variant carriers displays a specific metabolic signature. Methods: We applied a nontargeted clinical metabolomics pipeline based on ultra-high-pressure liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) allowing the exploration of 500 polar metabolites in plasma. We compared the plasma metabolic profiles of 25 patients with various OPA1 pathogenic variants and phenotypes to those of 20 healthy controls. Statistical analyses were performed using univariate and multivariate (principal component analysis [PCA], orthogonal partial least-squares discriminant analysis [OPLS-DA]) methods and a machine learning approach, the Biosigner algorithm. Results: A robust and relevant predictive model characterizing OPA1 individuals was obtained, based on a complex panel of metabolites with altered concentrations. An impairment of the purine metabolism, including significant differences in xanthine, hypoxanthine, and inosine concentrations, was at the foreground of this signature. In addition, the signature was characterized by differences in urocanate, choline, phosphocholine, glycerate, 1-oleoyl-rac-glycerol, rac-glycerol-1-myristate, aspartate, glutamate, and cystine concentrations. Conclusions: This first metabolic signature reported in the plasma of patient carrying OPA1 pathogenic variants highlights the unexpected involvement of purine metabolism in the pathophysiology of DOA.

Metabolomics and Lipidomics Profiling of a Combined Mitochondrial Plus Endoplasmic Reticulum Fraction of Human Fibroblasts: A Robust Tool for Clinical Studies.

Mitochondria and endoplasmic reticulum (ER) are physically and functionally connected. This close interaction, via mitochondria-associated membranes, is increasingly explored and supports the importance of studying these two organelles as a whole. Metabolomics and lipidomics are powerful approaches for the exploration of metabolic pathways that may be useful to provide deeper information on these organelles' functions, dysfunctions, and interactions. We developed a quick and simple experimental procedure for the purification of a mitochondria-ER fraction from human fibroblasts. We applied combined metabolomics and lipidomics analyses by mass spectrometry with excellent reproducibility. Seventy-two metabolites and 418 complex lipids were detected with a mean coefficient of variation around 12%, among which many were specific to the mitochondrial metabolism. Thus this strategy based on robust mitochondria-ER extraction and "omics" combination will be useful for investigating the pathophysiology of complex diseases.

A Nontargeted UHPLC-HRMS Metabolomics Pipeline for Metabolite Identification: Application to Cardiac Remote Ischemic Preconditioning.

In recent years, the number of investigations based on nontargeted metabolomics has increased, although often without a thorough assessment of analytical strategies applied to acquire data. Following published guidelines for metabolomics experiments, we report a validated nontargeted metabolomics strategy with pipeline for unequivocal identification of metabolites using the MSMLS molecule library. We achieved an in-house database containing accurate m/z values, retention times, isotopic patterns, full MS, and MS/MS spectra. A UHPLC-HRMS Q-Exactive method was developed, and experimental variations were determined within and between 3 experimental days. The extraction efficiency as well as the accuracy, precision, repeatability, and linearity of the method were assessed, the method demonstrating good performances. The methodology was further blindly applied to plasma from remote ischemic pre-conditioning (RIPC) rats. Samples, previously analyzed by targeted metabolomics using completely different protocol, analytical strategy, and platform, were submitted to our analytical pipeline. A combination of multivariate and univariate statistical analyses was employed. Selection of putative biomarkers from OPLS-DA model and S-plot was combined to jack-knife confidence intervals, metabolites' VIP values, and univariate statistics. Only variables with strong model contribution and highly statistical reliability were selected as discriminated metabolites. Three biomarkers identified by the previous targeted metabolomics study were found in the current work, in addition to three novel metabolites, emphasizing the efficiency of the current methodology and its ability to identify new biomarkers of clinical interest, in a single sequence. The biomarkers were identified to level 1 according to the metabolomics standard initiative and confirmed by both RPLC and HILIC-HRMS.

The metabolomic signature of Leber's hereditary optic neuropathy reveals endoplasmic reticulum stress.

Leber's hereditary optic neuropathy (MIM#535000), the commonest mitochondrial DNA-related disease, is caused by mutations affecting mitochondrial complex I. The clinical expression of the disorder, usually occurring in young adults, is typically characterized by subacute, usually sequential, bilateral visual loss, resulting from the degeneration of retinal ganglion cells. As the precise action of mitochondrial DNA mutations on the overall cell metabolism in Leber's hereditary optic neuropathy is unknown, we investigated the metabolomic profile of the disease. High performance liquid chromatography coupled with tandem mass spectrometry was used to quantify 188 metabolites in fibroblasts from 16 patients with Leber's hereditary optic neuropathy and eight healthy control subjects. Latent variable-based statistical methods were used to identify discriminating metabolites. One hundred and twenty-four of the metabolites were considered to be accurately quantified. A supervised orthogonal partial least squares discriminant analysis model separating patients with Leber's hereditary optic neuropathy from control subjects showed good predictive capability (Q 2cumulated = 0.57). Thirty-eight metabolites appeared to be the most significant variables, defining a Leber's hereditary optic neuropathy metabolic signature that revealed decreased concentrations of all proteinogenic amino acids, spermidine, putrescine, isovaleryl-carnitine, propionyl-carnitine and five sphingomyelin species, together with increased concentrations of 10 phosphatidylcholine species. This signature was not reproduced by the inhibition of complex I with rotenone or piericidin A in control fibroblasts. The importance of sphingomyelins and phosphatidylcholines in the Leber's hereditary optic neuropathy signature, together with the decreased amino acid pool, suggested an involvement of the endoplasmic reticulum. This was confirmed by the significantly increased phosphorylation of PERK and eIF2α, as well as the greater expression of C/EBP homologous protein and the increased XBP1 splicing, in fibroblasts from affected patients, all these changes being reversed by the endoplasmic reticulum stress inhibitor, TUDCA (tauroursodeoxycholic acid). Thus, our metabolomic analysis reveals a pharmacologically-reversible endoplasmic reticulum stress in complex I-related Leber's hereditary optic neuropathy fibroblasts, a finding that may open up new therapeutic perspectives for the treatment of Leber's hereditary optic neuropathy with endoplasmic reticulum-targeting drugs.