Plasma based targeted metabolomic analysis reveals alterations of phosphatidylcholines and oxidative stress markers in guinea pig model of allergic asthma

Bronchial asthma is one of the most common, chronic respiratory diseases, characterized by reversible airway obstruction, eosinophil and Th2 infiltration, airway hyperresponsiveness and airway remodelling; with many cells and mediators involved. Metabolomics is a relatively new field in “omics” sciences enabling the identification of metabolome for better diagnostics and studying of diseases phenotype.The aim of this study was to investigate the role of targeted metabolomics study for better understanding of the bronchial asthma pathophysiology and finding potential biomarkers in experimental models of eosinophilic inflammation.Plasma level of 185 metabolites was measured with the AbsoluteIDQ™ p180 kit in guinea pigs with experimentally-induced allergic inflammation (n = 15) compared to naïve non-sensitised and non-challenged controls (n = 18).Of the 185 metabolites identified in plasma, 22 were significantly different and changed in ovalbumin sensitised animals. Plasma level of 13 phosphatidylcholines with saturated and unsaturated long-chain fatty acids, total phosphatidylcholines count, carnitine, symmetric dimethylarginine and its ratio to total unmodified arginine, and kynurenine to tryptophan ratio were found to be decreased, while phospholipase A2 activity indicator, tryptophan, taurine and ratio of methionine sulfoxide to unmodified methionine were found to be increased in sensitised guinea pigs compared to naïve controls.Targeted metabolomic analysis revealed significant differences in plasma metabolome of sensitised guinea pigs. Our observations point to the activation of inflammatory and immune pathways, as well as the involvement of oxidative stress.     

Automated behavioral phenotyping reveals presymptomatic alterations in a SCA3 genetrap mouse model

Characterization of disease models of neurodegenerative disorders requires a systematic and comprehensive phenotyping in a highly standardized manner.Therefore,automated high-resolution behavior test systems such as the homecage based LabMaster system are of particular interest.We demonstrate the power of the automated LabMaster system by discovering previously unrecognized features of a recently characterized atxn3 mutant mouse model.This model provided neurological symptoms including gait ataxia,tremor,weight loss and premature death at the age of 12 months usually detectable just 2 weeks before the mice died.Moreover,using the LabMaster system we were able to detect hypoactivity in presymptomatic mutant mice in the dark as well as light phase.Additionally,we analyzed inflammation,immunological and hematological parameters,which indicated a reduced immune defense in phenotypic mice.Here we demonstrate that a detailed characterization even of organ systems that are usually not affected in SCA3 is important for further studies of pathogenesis and required for the preclinical therapeutic studies.     

Proteomic analysis of colorectal cancer reveals alterations in metabolic pathways: mechanism of tumorigenesis

Colorectal cancer is the second leading killer cancer worldwide and presently the most common cancer among males in Singapore. The study aimed to detect changes of protein profiles associated with the process of colorectal tumorigenesis to identify specific protein markers for early colorectal cancer detection and diagnosis or as potential therapeutic targets. Seven pairs of colorectal cancer tissues and adjacent normal mucosa were examined by two-dimensional gel electrophoresis at basic pH range (pH 7-10). Intensity changes of 34 spots were detected with statistical significance. 16 of the 34 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels revealed a significantly enhanced glycolytic pathway (Warburg effect), a decreased gluconeogenesis, a suppressed glucuronic acid pathway, and an impaired tricarboxylic acid cycle. Observed changes in protein abundance were verified by two-dimensional DIGE. These changes reveal an underlying mechanism of colorectal tumorigenesis in which the roles of impaired tricarboxylic acid cycle and the Warburg effect may be critical.     

Analysis of the fibroblast growth factor system reveals alterations in a mouse model of spinal muscular atrophy

The monogenetic disease Spinal Muscular Atrophy (SMA) is characterized by a progressive loss of motoneurons leading to muscle weakness and atrophy due to severe reduction of the Survival of Motoneuron (SMN) protein. Several models of SMA show deficits in neurite outgrowth and maintenance of neuromuscular junction (NMJ) structure. Survival of motoneurons, axonal outgrowth and formation of NMJ is controlled by neurotrophic factors such as the Fibroblast Growth Factor (FGF) system. Besides their classical role as extracellular ligands, some FGFs exert also intracellular functions controlling neuronal differentiation. We have previously shown that intracellular FGF-2 binds to SMN and regulates the number of a subtype of nuclear bodies which are reduced in SMA patients. In the light of these findings, we systematically analyzed the FGF-system comprising five canonical receptors and 22 ligands in a severe mouse model of SMA. In this study, we demonstrate widespread alterations of the FGF-system in both muscle and spinal cord. Importantly, FGF-receptor 1 is upregulated in spinal cord at a pre-symptomatic stage as well as in a mouse motoneuron-like cell-line NSC34 based model of SMA. Consistent with that, phosphorylations of FGFR-downstream targets Akt and ERK are increased. Moreover, ERK hyper-phosphorylation is functionally linked to FGFR-1 as revealed by receptor inhibition experiments. Our study shows that the FGF system is dysregulated at an early stage in SMA and may contribute to the SMA pathogenesis.     

Cognitive and emotional alterations are related to hippocampal inflammation in a mouse model of metabolic syndrome

Converging clinical data suggest that peripheral inflammation is likely involved in the pathogenesis of the neuropsychiatric symptoms associated with metabolic syndrome (MetS). However, the question arises as to whether the increased prevalence of behavioral alterations in MetS is also associated with central inflammation, i.e. cytokine activation, in brain areas particularly involved in controlling behavior. To answer this question, we measured in a mouse model of MetS, namely the diabetic and obese db/db mice, and in their healthy db/+ littermates emotional behaviors and memory performances, as well as plasma levels and brain expression (hippocampus; hypothalamus) of inflammatory cytokines. Our results shows that db/db mice displayed increased anxiety-like behaviors in the open-field and the elevated plus-maze (i.e. reduced percent of time spent in anxiogenic areas of each device), but not depressive-like behaviors as assessed by immobility time in the forced swim and tail suspension tests. Moreover, db/db mice displayed impaired spatial recognition memory (hippocampus-dependent task), but unaltered object recognition memory (hippocampus-independent task). In agreement with the well-established role of the hippocampus in anxiety-like behavior and spatial memory, behavioral alterations of db/db mice were associated with increased inflammatory cytokines (interleukin-1β, tumor necrosis factor-α and interleukin-6) and reduced expression of brain-derived neurotrophic factor (BDNF) in the hippocampus but not the hypothalamus. These results strongly point to interactions between cytokines and central processes involving the hippocampus as important contributing factor to the behavioral alterations of db/db mice. These findings may prove valuable for introducing novel approaches to treat neuropsychiatric complications associated with MetS.     

Comprehensive metabolomic analysis of peanut-induced anaphylaxis in a murine model

Acute anaphylaxis caused by food allergy is a potentially life-threatening allergic reaction that results in thousands of hospitalizations in North America every year. There are currently no effective means of treatment for anaphylaxis beyond the administration of epinephrine which is only given after a reaction has already started. Furthermore, knowledge regarding the underlying physico-chemical basis of anaphylaxis is largely incomplete. Arguably, the study of anaphylaxis by comprehensive metabolomics offers a unique opportunity to study this phenomenon due to its ability to provide a snap-shot of the chemical state of a subject experiencing anaphylaxis encompassing both endogenous and exogenous factors including dietary and environmental effects. In this study, we sought to apply a comprehensive metabolomics approach using liquid chromatography–mass spectrometry to a murine model of peanut-induced anaphylaxis. The results of this study revealed that the metabolomic profiles of mice experiencing peanut-induced anaphylaxis followed a distinct pattern that mirrors the time-stage of anaphylaxis being experienced. Direct comparison of anaphylactic mice to time-matched control mice showed that the metabolomic profiles changed considerably as the anaphylactic reaction progressed. This comparison also revealed changes in both known biomarkers of anaphylaxis such as histamine and methylhistamine as well as a suite of metabolites chemically similar to known anaphylaxis mediators but whose biological functions are not well understood. These metabolites include a number of phospholipids such as a variety of phosphatidylcholines and lyso-phosphatidylcholines that may be of interest for further investigation regarding their potential as anaphylaxis mediators or chemical precursors to mediators.     

Early onset of craniosynostosis in an Apert mouse model reveals critical features of this pathology

Activating mutations of FGFRs1-3 cause craniosynostosis (CS), the premature fusion of cranial bones, in man and mouse. The mechanisms by which such mutations lead to CS have been variously ascribed to increased osteoblast proliferation, differentiation, and apoptosis, but it is not always clear how these disturbances relate to the process of suture fusion. We have reassessed coronal suture fusion in an Apert Fgfr2 (S252W) mouse model. We find that the critical event of CS is the early loss of basal sutural mesenchyme as the osteogenic fronts, expressing activated Fgfr2, unite to form a contiguous skeletogenic membrane. A mild increase in osteoprogenitor proliferation precedes but does not accompany this event, and apoptosis is insignificant. On the other hand, the more apical coronal suture initially forms appropriately but then undergoes fusion, albeit at a slower rate, accompanied by a significant decrease in osteoprogenitor proliferation, and increased osteoblast maturation. Apoptosis now accompanies fusion, but is restricted to bone fronts in contact with one another. We correlated these in vivo observations with the intrinsic effects of the activated Fgfr2 S252W mutation in primary osteoblasts in culture, which show an increased capacity for both proliferation and differentiation. Our studies suggest that the major determinant of Fgfr2-induced craniosynostosis is the failure to respond to signals that would halt the recruitment or the advancement of osteoprogenitor cells at the sites where sutures should normally form.     

Nontargeted metabolomic analysis of skeletal muscle in a dehydroepiandrosterone‐induced mouse model of polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy and an important metabolic disorder in women of reproductive age. Insulin resistance (IR) is one of its most important clinical features in patients with PCOS. Androgen excess‐induced mitochondrial dysfunction contributes to skeletal muscle IR in dehydroepiandrosterone (DHEA)‐induced PCOS mice. The effect of androgen excess on the skeletal muscle, however, is incompletely characterized. A nontargeted metabolomics approach was thus applied to analyze the metabolites in skeletal muscle of DHEA‐induced PCOS mice. Data from metabolomic analysis revealed the significant changes in 32 metabolites and the marked impact of five metabolic pathways. ATP production was also found to be significantly reduced in skeletal muscle of DHEA mice. Combined with the quantification of type I and II myofibers and lipid measurement in the skeletal muscle of the mice, the results from the present study supported the role of mitochondrial impairment rather than lipid accumulation in the pathogenesis of skeletal muscle IR in DHEA‐induced PCOS mice. In summary, we show here for the first time the profile of the metabolites in the skeletal muscle of DHEA‐induced PCOS mice which exhibit IR. The work would help better understand the pathology of skeletal muscle IR in PCOS.     

Analysis of the cerebellar proteome in a transgenic mouse model of inherited prion disease reveals preclinical alteration of calcineurin activity

Inherited prion diseases are linked to insertional and point mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. Transgenic (Tg) (PG14) mice express a mouse PrP homolog of a nine-octapeptide insertion associated with an inherited prion disorder. These mice develop a progressive neurological syndrome characterized by ataxia and cerebellar atrophy due to synaptic degeneration in the molecular layer and massive apoptosis of granule neurons. To investigate the molecular events that may contribute to neurological dysfunction, we carried out a differential proteomic analysis of cerebella from Tg(PG14) mice at the preclinical, onset, and symptomatic phases of their neurological illness. 2-D maps of cerebellar proteins from Tg(PG14) mice were compared to those obtained from age-matched Tg(WT) mice that express wild-type PrP and remain healthy. Proteins whose levels were significantly modified in at least one stage of the Tg(PG14) disease were identified by PMF. Analysis detected a preclinical decrease of the calcium/calmodulin-dependent phosphatase calcineurin (CaN) in granule neurons, suggesting that dysregulation of CaN activity induced by mutant PrP may be responsible for the cerebellar dysfunction in Tg(PG14) mice.     

Optimized proteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags

Recent proteomic applications have demonstrated their potential for revealing the molecular mechanisms underlying neurodegeneration. The present study quantifies cerebellar protein changes in mice that are deficient in plasma membrane calcium ATPase 2 (PMCA2), an essential neuronal pump that extrudes calcium from cells and is abundantly expressed in Purkinje neurons. PMCA2-null mice display motor dyscoordination and unsteady gait deficits observed in neurological diseases such as multiple sclerosis and ataxia. We optimized an amine-specific isobaric tags (iTRAQ)-based shotgun proteomics workflow for this study. This workflow took consideration of analytical variance as a function of ion signal intensity and employed biological repeats to aid noise reduction. Even with stringent protein identification criteria, we could reliably quantify nearly 1000 proteins, including many neuronal proteins that are important for synaptic function. We identified 21 proteins that were differentially expressed in PMCA2-null mice. These proteins are involved in calcium homeostasis, cell structure and chromosome organization. Our findings shed light on the molecular changes that underlie the neurological deficits observed in PMCA2-null mice. The optimized workflow presented here will be valuable for others who plan to implement the iTRAQ method.     

Acute renal proximal tubule alterations during induced metabolic crises in a mouse model of glutaric aciduria type 1

The metabolic disorder glutaric aciduria type 1 (GA1) is caused by deficiency of the mitochondrial glutaryl-CoA dehydrogenase (GCDH), leading to accumulation of the pathologic metabolites glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in blood, urine and tissues. Affected patients are prone to metabolic crises developing during catabolic conditions, with an irreversible destruction of striatal neurons and a subsequent dystonic–dyskinetic movement disorder. The pathogenetic mechanisms mediated by GA and 3OHGA have not been fully characterized. Recently, we have shown that GA and 3OHGA are translocated through membranes via sodium-dependent dicarboxylate cotransporter (NaC) 3, and organic anion transporters (OATs) 1 and 4. Here, we show that induced metabolic crises in Gcdh^?/? mice lead to an altered renal expression pattern of NaC3 and OATs, and the subsequent intracellular GA and 3OHGA accumulation. Furthermore, OAT1 transporters are mislocalized to the apical membrane during metabolic crises accompanied by a pronounced thinning of proximal tubule brush border membranes. Moreover, mitochondrial swelling and increased excretion of low molecular weight proteins indicate functional tubulopathy. As the data clearly demonstrate renal proximal tubule alterations in this GA1 mouse model during induced metabolic crises, we propose careful evaluation of renal function in GA1 patients, particularly during acute crises. Further studies are needed to investigate if these findings can be confirmed in humans, especially in the long-term outcome of affected patients.     

Proteomic and metabolomic profiling of a trait anxiety mouse model implicate affected pathways

Depression and anxiety disorders affect a great number of people worldwide. Whereas singular factors have been associated with the pathogenesis of psychiatric disorders, growing evidence emphasizes the significance of dysfunctional neural circuits and signaling pathways. Hence, a systems biology approach is required to get a better understanding of psychiatric phenotypes such as depression and anxiety. Furthermore, the availability of biomarkers for these disorders is critical for improved diagnosis and monitoring treatment response. In the present study, a mouse model presenting with robust high versus low anxiety phenotypes was subjected to thorough molecular biomarker and pathway discovery analyses. Reference animals were metabolically labeled with the stable (15)N isotope allowing an accurate comparison of protein expression levels between the high anxiety-related behavior versus low anxiety-related behavior mouse lines using quantitative mass spectrometry. Plasma metabolomic analyses identified a number of small molecule biomarkers characteristic for the anxiety phenotype with particular focus on myo-inositol and glutamate as well as the intermediates involved in the tricarboxylic acid cycle. In silico analyses suggested pathways and subnetworks as relevant for the anxiety phenotype. Our data demonstrate that the high anxiety-related behavior and low anxiety-related behavior mouse model is a valuable tool for anxiety disorder drug discovery efforts.     

Lung proteome alterations in a mouse model for nonallergic asthma

A mouse model for nonatopic asthma was employed to study the alterations of the lung proteome to gain insight into the underlying molecular mechanisms of disease pathophysiology post-challenge. Lung samples from asthmatic and control mice were used to generate 24 high quality two-dimensional electrophoresis gels wherein 2115 proteins were examined for disease relevance. In total, 23 proteins were significantly up- or down-regulated following hapten-challenge of dinitro-fluorobenzene-hypersensitive mice. Twenty proteins were identified by mass spectrometry, of which 18 could be linked to asthma related symptoms, such as stress and inflammation, lung detoxification, plasma exudation and/or tissue remodeling. As such, proteomics was clearly vindicated as a means of studying this complex disease phenomenon. The proteins found in this study may not necessarily play a role in the immunological mechanisms and/or pathophysiology of asthma development. However, they may prove useful as surrogate biomarkers for quantitatively monitoring disease state progression or response to therapy. The mathematics of achieving statistical confidence from low numbers of gel replicates containing large numbers of independent variables stress the need for high numbers of replicates to better sample the population of proteins revealed by two-dimensional gel electrophoresis.     

Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia

Polyglutamine disorders are inherited neurodegenerative diseases caused by the accumulation of expanded polyglutamine protein (polyQ). Previously, we identified a new guanosine triphosphatase, CRAG, which facilitates the degradation of polyQ aggregates through the ubiquitin-proteasome pathway in cultured cells. Because expression of CRAG decreases in the adult brain, a reduced level of CRAG could underlie the onset of polyglutamine diseases. To examine the potential of CRAG expression for treating polyglutamine diseases, we generated model mice expressing polyQ predominantly in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. Lentivector-mediated expression of CRAG in Purkinje cells of model mice extensively cleared polyQ aggregates and re-activated dendritic differentiation, resulting in a striking rescue from ataxia. Our in vivo data substantiate previous cell-culture-based results and extend further the usefulness of targeted delivery of CRAG as a gene therapy for polyglutamine diseases.     

Defective Wnt-dependent cerebellar midline fusion in a mouse model of Joubert syndrome

The ciliopathy Joubert syndrome is marked by cerebellar vermis hypoplasia, a phenotype for which the pathogenic mechanism is unclear. To investigate Joubert syndrome pathogenesis, we have examined mice with mutated Ahi1, the first identified Joubert syndrome-associated gene. These mice show cerebellar hypoplasia with a vermis-midline fusion defect early in development. This defect is concomitant with expansion of the roof plate and is also evident in a mouse mutant for another Joubert syndrome-associated gene, Cep290. Furthermore, fetal magnetic resonance imaging (MRI) of human subjects with Joubert syndrome reveals a similar midline cleft, suggesting parallel pathogenic mechanisms. Previous evidence has suggested a role for Jouberin (Jbn), the protein encoded by Ahi1, in canonical Wnt signaling. Consistent with this, we found decreased Wnt reporter activity at the site of hemisphere fusion in the developing cerebellum of Ahi1-mutant mice. This decrease was accompanied by reduced proliferation at the site of fusion. Finally, treatment with lithium, a Wnt pathway agonist, partially rescued this phenotype. Our findings implicate a defect in Wnt signaling in the cerebellar midline phenotype seen in Joubert syndrome that can be overcome with Wnt stimulation.