Hamid Suhail

AMP-Activated Protein Kinase Suppresses Autoimmune Central Nervous System Disease by Regulating M1-Type Macrophage-Th17 Axis.

The AMP-activated protein kinase, AMPK, is an energy-sensing, metabolic switch implicated in various metabolic disorders; however, its role in inflammation is not well defined. We have previously shown that loss of AMPK exacerbates experimental autoimmune encephalomyelitis (EAE) disease severity. In this study, we investigated the mechanism through which AMPK modulates inflammatory disease like EAE. AMPKα1 knockout (α1KO) mice with EAE showed severe demyelination and inflammation in the brain and spinal cord compared with wild-type due to higher expression of proinflammatory Th17 cytokines, including IL-17, IL-23, and IL-1β, impaired blood–brain barrier integrity, and increased infiltration of inflammatory cells in the CNS. Infiltrated CD4 cells in the brains and spinal cords of α1KO with EAE were significantly higher compared with wild-type EAE and were characterized as IL-17 (IL-17 and GM-CSF double-positive) CD4 cells. Increased inflammatory response in α1KO mice was due to polarization of macrophages (Mϕ) to proinflammatory M1 type phenotype (IL-10lowIL-23/IL-1β/IL-6high), and these M1 Mϕ showed stronger capacity to induce allogenic as well as Ag-specific (myelin oligodendrocyte glycoprotein [MOG]35–55) T cell response. Mϕ from α1KO mice also enhanced the encephalitogenic property of MOG35–55–primed CD4 T cells in B6 mice. The increased encephalitogenic MOG-restricted CD4+ T cells were due to an autocrine effect of IL-1β/IL-23–mediated induction of IL-6 production in α1KO Mϕ, which in turn induce IL-17 and GM-CSF production in CD4 cells. Collectively, our data indicate that AMPK controls the inflammatory disease by regulating the M1 phenotype–Th17 axis in an animal model of multiple sclerosis.

Untargeted plasma metabolomics identifies endogenous metabolite with drug-like properties in chronic animal model of multiple sclerosis

We performed untargeted metabolomics in plasma of B6 mice with experimental autoimmune encephalitis (EAE) at the chronic phase of the disease in search of an altered metabolic pathway(s). Of 324 metabolites measured, 100 metabolites that mapped to various pathways (mainly lipids) linked to mitochondrial function, inflammation, and membrane stability were observed to be significantly altered between EAE and control (p < 0.05, false discovery rate <0.10). Bioinformatics analysis revealed six metabolic pathways being impacted and altered in EAE, including α-linolenic acid and linoleic acid metabolism (PUFA). The metabolites of PUFAs, including ω-3 and ω-6 fatty acids, are commonly decreased in mouse models of multiple sclerosis (MS) and in patients with MS. Daily oral administration of resolvin D1, a downstream metabolite of ω-3, decreased disease progression by suppressing autoreactive T cells and inducing an M2 phenotype of monocytes/macrophages and resident brain microglial cells. This study provides a proof of principle for the application of metabolomics to identify an endogenous metabolite(s) possessing drug-like properties, which is assessed for therapy in preclinical mouse models of MS.

Metformin‐induced mitochondrial function and ABCD2 up‐regulation in X‐linked adrenoleukodystrophy involves AMP‐activated protein kinase

X‐linked adrenoleukodystrophy (X‐ALD) is a progressive neurometabolic disease caused by mutations/deletions in the Abcd1 gene. Similar mutations/deletions in the Abcd1 gene often result in diagonally opposing phenotypes of mild adrenomyeloneuropathy and severe neuroinflammatory cerebral adrenoleukodystrophy (ALD), which suggests involvement of downstream modifier genes. We recently documented the first evidence of loss of AMP‐activated protein kinase α1 (AMPKα1) in ALD patient‐derived cells. Here, we report the novel loss of AMPKα1 in postmortem brain white matter of patients with ALD phenotype. Pharmacological activation of AMPK can rescue the mitochondrial dysfunction and inhibit the pro‐inflammatory response. The FDA approved anti‐diabetic drug Metformin, a well‐known AMPK activator, induces mitochondrial biogenesis and is documented for its anti‐inflammatory role. We observed a dose‐dependent activation of AMPKα1 in metformin‐treated X‐ALD patient‐derived fibroblasts. Metformin also induced mitochondrial oxidative phosphorylation and ATP levels in X‐ALD patient‐derived fibroblasts. Metformin treatment decreased very long chain fatty acid levels and pro‐inflammatory cytokine gene expressions in X‐ALD patient‐derived cells. Abcd2 [adrenoleukodystrophy protein‐related protein] levels were increased in metformin‐treated X‐ALD patient‐derived fibroblasts and Abcd1‐KO mice primary mixed glial cells. Abcd2 induction was AMPKα1‐dependent since metformin failed to induce Abcd2 levels in AMPKα1‐KO mice‐derived primary mixed glial cells. In vivo metformin (100 mg/Kg) in drinking water for 60 days induced Abcd2 levels and mitochondrial oxidative phosphorylation protein levels in the brain and spinal cord of Abcd1‐KO mice. Taken together, these results provide proof‐of‐principle for therapeutic potential of metformin as a useful strategy for correcting the metabolic and inflammatory derangements in X‐ALD by targeting AMPK.

Loss of AMP-Activated Protein Kinase Induces Mitochondrial Dysfunction and Proinflammatory Response in Unstimulated Abcd1-Knockout Mice Mixed Glial Cells

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations and/or deletions in the ABCD1 gene. Similar mutations/deletions can give rise to variable phenotypes ranging from mild adrenomyeloneuropathy (AMN) to inflammatory fatal cerebral adrenoleukodystrophy (ALD) via unknown mechanisms. We recently reported the loss of the anti-inflammatory protein adenosine monophosphate activated protein kinase (AMPKα1) exclusively in ALD patient-derived cells. X-ALD mouse model (Abcd1-knockout (KO) mice) mimics the human AMN phenotype and does not develop the cerebral inflammation characteristic of human ALD. In this study we document that AMPKα1 levels in vivo (in brain cortex and spinal cord) and in vitro in Abcd1-KO mixed glial cells are similar to that of wild type mice. Deletion of AMPKα1 in the mixed glial cells of Abcd1-KO mice induced spontaneous mitochondrial dysfunction (lower oxygen consumption rate and ATP levels). Mitochondrial dysfunction in ALD patient-derived cells and in AMPKα1-deleted Abcd1-KO mice mixed glial cells was accompanied by lower levels of mitochondrial complex (1-V) subunits. More importantly, AMPKα1 deletion induced proinflammatory inducible nitric oxide synthase levels in the unstimulated Abcd1-KO mice mixed glial cells. Taken together, this study provides novel direct evidence for a causal role for AMPK loss in the development of mitochondrial dysfunction and proinflammatory response in X-ALD.

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

AbstractIdentification 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 AbstractUntargeted 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.

Analysis of stable isotope assisted metabolomics data acquired by GC-MS

Stable isotope assisted metabolomics (SIAM) measures the abundance levels of metabolites in a particular pathway using stable isotope tracers (e.g., 13C, 18O and/or 15N). We report a method termed signature ion approach for analysis of SIAM data acquired on a GC-MS system equipped with an electron ionization (EI) ion source. The signature ion is a fragment ion in EI mass spectrum of a derivatized metabolite that contains all atoms of the underivatized metabolite, except the hydrogen atoms lost during derivatization. In this approach, GC-MS data of metabolite standards were used to recognize the signature ion from the EI mass spectra acquired from stable isotope labeled samples, and a linear regression model was used to deconvolute the intensity of overlapping isotopologues. A mixture score function was also employed for cross-sample chromatographic peak list alignment to recognize the chromatographic peaks generated by the same metabolite in different samples, by simultaneously evaluating the similarity of retention time and EI mass spectrum of two chromatographic peaks. Analysis of a mixture of 16 13C-labeled and 16 unlabeled amino acids showed that the signature ion approach accurately identified and quantified all isotopologues. Analysis of polar metabolite extracts from cells respectively fed with uniform 13C-glucose and 13C-glutamine further demonstrated that this method can also be used to analyze the complex data acquired from biological samples.

MicroRNA Regulation of Proinflammatory Response in X-linked Adrenoleukodystrophy

The underlying mechanism(s) for development of the inflammatory response in inherited, fatal neurometabolic disease X-linked adrenoleukodystrophy (X-ALD) remain completely unknown. Genetic defect (ABCD1 mutation/ deletion), common to all phenotypes of X-ALD, has failed to explain the development of inflammation only in a subset of patients. In this study we document the novel role of microRNAs (miRNAs) in the development of the inflammatory response in unstimulated ALD patient-derived lymphocytes and Abcd1-knockout (Abcd1-KO) mice mixed glial cells. The levels of proinflammatory cytokine gene expression (inducible nitric oxide synthase [iNOS]) were increased in X-ALD patient-derived lymphocytes. Predictions via the use of online bioinformatics algorithms and confirmed by using miRNA mimic of inhibitor-transfection method (gain- and loss-of-function) revealed the role of miR-323-5p in regulating iNOS expression in X-ALD patient-derived lymphocytes. Functional confirmation of the targets was obtained by using the dual-luciferase assay and western blot analysis. Abcd1-KO mice do not develop the inflammatory response characteristic of the fatal X-ALD phenotype. We recently reported that AMP-activated protein kinase (AMPKα1) deletion induced spontaneous iNOS expression in Abcd1-KO mice mixed glial cells. Here we discover the novel role of miR-323-5p regulating the iNOS response in AMPKα1-deleted Abcd1-KO mice mixed glial cells. This study demonstrated the novel role of miR-323-5p in regulating the inflammatory response in unstimulated X-ALD patient-derived cells and mixed glial cells from Abcd1-KO mice suggesting that these miRNA could function as promising novel therapeutic targets for the treatment of X-ALD.