Urinary and Plasma Metabolomics Identify the Distinct Metabolic Profile of Disease State in Chronic Mouse Model of Multiple Sclerosis.

Identification of non-invasive biomarkers of disease progression in multiple sclerosis (MS) is critically needed for monitoring the disease progression and for effective therapeutic interventions. Urine is an attractive source for non-invasive biomarkers because it is easily obtained in the clinic. In search of a urine metabolite signature of progression in chronic experimental autoimmune encephalomyelitis (EAE), we profiled urine at the chronic stage of the disease (day 45 post immunization) by global untargeted metabolomics. Using a combination of high-throughput liquid-and-gas chromatography with mass spectrometry, we found 105 metabolites (P < 0.05) significantly altered at the chronic stage, indicating a robust alteration in the urine metabolite profile during disease. Assessment of altered metabolites against the Kyoto Encyclopedia of Genes and Genomes revealed distinct non-overlapping metabolic pathways and revealed phenylalanine-tyrosine and associated metabolism being the most impacted. Combined with previously performed plasma profiling, eight common metabolites were significantly altered in both of the biofluids. Metaboanalyst analysis of these common metabolites revealed that phenylalanine metabolism and Valine, leucine, and isoleucine biosynthetic pathways are central metabolic pathways in both bio-fluids and could be analyzed further, either for the discovery of therapeutics or biomarker development. Overall, our study suggests that urine and plasma metabolomics may contribute to the identification of a distinct metabolic fingerprint of EAE disease discriminating from the healthy control which may aid in the development of an objective non-invasive monitoring method for progressive autoimmune diseases like MS. Graphical Abstract Untargeted urinary metabolomics of a chronic mouse model of multiple sclerosis identified Phenylalanine, tyrosine & tryptophan metabolism as the significantly altered metabolic pathway. Eight common metabolites were identified when we combined urinary and plasma metabolic signature, which revealed a perturbation of Phenylalanine metabolism and valine, leucine & isoleucine metabolic pathways, involved in CNS dysfunction during diseases. The identified eight metabolic signature of urine and plasma may be of clinical relevance as potential biomarkers and guide towards the identification of specific metabolic pathways as novel drug targets.

Targeted Stage-Specific Inflammatory microRNA Profiling in Urine During Disease Progression in Experimental Autoimmune Encephalomyelitis: Markers of Disease Progression and Drug Response.

Recently, microRNAs (miRNAs) have been implicated in regulating neuroinflammatory and demyelinative responses in multiple sclerosis (MS) and its mouse model of experimental autoimmune encephalomyelitis (EAE). miRNAs have also been studied as biomarkers of disease pathology and drug-response in MS. However, no complete miRNA profiling at various stages of EAE disease has been examined, especially in the urine. We carried out a systematic analysis of miRNAs in the urine exosomes as well as in the plasma and spinal cord at pre-onset, onset and peak stages of EAE established in the chronic B6 mice model. For the first time, we provide evidence that urine exosomes can be a specific and sensitive source of miRNA biomarkers for all 3 stages of EAE disease. In a significant observation, we observed that miR-155-5p expression increased in urine exosomes, plasma and spinal cord 6 days before the onset of disease, suggesting its early involvement in the pathology of EAE disease. We also analyzed the effect of Glatiramer acetate (GA; copaxone) treatment, an approved treatment for MS patients, in modulating miRNA expression at the peak of EAE disease. We identified miR-155-5p, miR-27a-3p, miR-9-5p and miR-350-5p as putative GA-treatment responsive miRNA biomarkers. Since, EAE is a mainly CD4 cells mediated disease, we also examined the above set of miRNAs and found to be significantly altered in T cells polarized to Th1 and Th17 phenotype, similar to urine exosomes. Thus, urine exosome miRNAs hold the potential to be defined as novel accessible stage-specific biomarkers of EAE (MS) disease as well as treatment response.

NMR-Based Metabolomics Separates the Distinct Stages of Disease in a Chronic Relapsing Model of Multiple Sclerosis.

Relapsing experimental allergic encephalomyelitis (Cr-EAE) is commonly used to explore the pathogenesis and efficacy of new therapies for MS, but it is unclear whether the metabolome of Cr-EAE is comparable to human multiple sclerosis (MS). For MS, the diagnosis and staging can be achieved by metabolomics on blood using a combination of magnetic resonance spectroscopy and partial least squares discriminant analysis (PLS-DA). Here, we sought to discover whether this approach could be used to differentiate between sequential disease states in Cr-EAE and whether the same metabolites would be discriminatory. Urine and plasma samples were obtained at different time-points from a clinically relevant model of MS. Using PLS-DA modelling for the urine samples furnished some predictive models, but could not discriminate between all disease states. However, PLS-DA modelling of the plasma samples was able to distinguish between animals with clinically silent disease (day 10, 28) and animals with active disease (day 14, 38). We were also able to distinguish Cr-EAE mice from naive mice at all-time points and control mice, treated with complete Freund's adjuvant alone, at day 14 and 38. Key metabolites that underpin these models included fatty acids, glucose and taurine. Two of these metabolites, fatty acids and glucose, were also key metabolites in separating relapsing-remitting MS from secondary-progressive MS in the human study. These results demonstrate the sensitivity of this metabolomics approach for distinguishing between different disease states. Furthermore, some, but not all, of the changes in metabolites were conserved in humans and the mouse model, which could be useful for future drug development.

Early-life exposure to lipopolysaccharide reduces the severity of experimental autoimmune encephalomyelitis in adulthood and correlated with increased urine corticosterone and apoptotic CD4+ T cells.

Early-life exposure to bacterial endotoxins, such as lipopolysaccharides (LPS), can provide neuroprotection against experimental autoimmune encephalomyelitis (EAE) during adulthood, possibly through altering the responsiveness of the immune system. Here, we show that exposure of LPS to neonatal rats resulted in a sustained elevation of corticosterone level in urine when compared with saline-treated rats, and that the high level of urine corticosterone was maintained during the progression of EAE (P<0.05). This high level of production of corticosterone plays an important role in altering the predisposition to EAE-induced neuroinflammation, as a positive correlation occurred between the concentration of urine corticosterone and the increased apoptotic CD4(+) T cells from the peripheral blood. LPS-treated rats also showed a reduced number of CD3(+) T cells in the spinal cord. The splenic antigen-presenting cells showed a reduced expression of MHC II during EAE development in LPS-exposed rats when compared with rats exposed to the saline-treated control. Together, these findings suggest that treating neonatal rats with LPS evokes a sustained elevation of glucocorticoid, which may suppress immune response during EAE by increasing apoptosis of CD4(+) T cells and reducing the expression of MHC II on antigen-presenting cells. Therefore, exposing neonates to bacterial endotoxin may further be developed as an immunization strategy to prevent human multiple sclerosis and other inflammatory brain diseases.

Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental autoimmune encephalomyelitis.

Multiple sclerosis is an inflammatory, neurodegenerative disease for which experimental autoimmune encephalomyelitis (EAE) is a model. Treatments with estrogens have been shown to decrease the severity of EAE through anti-inflammatory mechanisms. Here we investigated whether treatment with an estrogen receptor alpha (ERalpha) ligand could recapitulate the estrogen-mediated protection in clinical EAE. We then went on to examine both anti-inflammatory and neuroprotective mechanisms. EAE was induced in wild-type, ERalpha-, or ERbeta-deficient mice, and each was treated with the highly selective ERalpha agonist, propyl pyrazole triol, to determine the effect on clinical outcomes, as well as on inflammatory and neurodegenerative changes. ERalpha ligand treatment ameliorated clinical disease in both wild-type and ERbeta knock-out mice, but not in ERalpha knock-out mice, thereby demonstrating that the ERalpha ligand maintained ERalpha selectivity in vivo during disease. ERalpha ligand treatment also induced favorable changes in autoantigen-specific cytokine production in the peripheral immune system [decreased TNFalpha, interferon-gamma, and interleukin-6, with increased interleukin-5] and decreased CNS white matter inflammation and demyelination. Interestingly, decreased neuronal staining [NeuN+ (neuronal-specific nuclear protein)/beta3-tubulin+/Nissl], accompanied by increased immunolabeling of microglial/monocyte (Mac 3+) cells surrounding these abnormal neurons, was observed in gray matter of spinal cords of EAE mice at the earliest stage of clinical disease, 1-2 d after the onset of clinical signs. Treatment with either estradiol or the ERalpha ligand significantly reduced this gray matter pathology. In conclusion, treatment with an ERalpha ligand is highly selective in vivo, mediating both anti-inflammatory and neuroprotective effects in EAE.

1H-NMR spectroscopy combined with pattern recognition analysis reveals characteristic chemical patterns in urines of MS patients and non-human primates with MS-like disease.

Proton nuclear magnetic resonance (1H-NMR) spectroscopy in combination with pattern recognition techniques were used to investigate the composition of organic compounds in urines from patients with multiple sclerosis (MS), patients with other neurological diseases (OND) and healthy controls (H). Using a valid animal model of MS, namely the common marmoset (Callithrix jacchus) model of experimental autoimmune encephalomyelitis (EAE), the relation of disease progression and alteration of the urine composition was investigated. Urine samples were collected during different stages of EAE, either induced with whole human myelin or with the myelin protein MOG in complete adjuvant. The urine samples were analysed with 1H-NMR spectroscopy allowing simultaneous detection of an array of compounds. Spectral differences between urines from EAE-affected and healthy monkeys were assessed with multivariate analysis. Evidence is provided that development of EAE is associated with changes in the chemical composition of the urine, in particular of compounds with NMR peaks in the region of the spectrum between 0.5 and 3.50 ppm. In addition, we found preliminary evidence for differences between urines from MS, OND and H groups.

Central nervous system demyelinating diseases and increased release of cholesterol into the urinary system of rats.

The question of what happens to cholesterol in the adult central nervous system during its slow turnover has been addressed using rats with brain and spinal cord labeled with [4-14C]cholesterol upon intracerebral injection of labeled cholesterol into rats at 10-12 days of age. At six months after injection, 14C was found only in the brain and spinal cord and was slowly released via the rat's urine. When labeled rats were given demyelinating agents (triethyl tin chloride, hexachlorophene, sodium cyanide) and when experimental allergic encephalomyelitis was induced, a measurable increase in urinary 14C label above control levels was found. It was concluded that there is a direct relationship between the experimental demyelination induced and the increased release of cholesterol metabolites into urine. The study suggests that a clinical method could be developed to determine the rate of central nervous system demyelination by measuring the amount of urinary cholesterol metabolites.