Disease-related changes in the cerebrospinal fluid metabolome in amyotrophic lateral sclerosis detected by GC/TOFMS

Background/Aim

The changes in the cerebrospinal fluid (CSF) metabolome associated with the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) are poorly understood and earlier smaller studies have shown conflicting results. The metabolomic methodology is suitable for screening large cohorts of samples. Global metabolomics can be used for detecting changes of metabolite concentrations in samples of fluids such as CSF.

Methodology

Using gas chromatography coupled to mass spectrometry (GC/TOFMS) and multivariate statistical modeling, we simultaneously studied the metabolome signature of ∼120 small metabolites in the CSF of patients with ALS, stratified according to hereditary disposition and clinical subtypes of ALS in relation to controls.

Principal Findings

The study is the first to report data validated over two sub-sets of ALS vs. control patients for a large set of metabolites analyzed by GC/TOFMS. We find that patients with sporadic amyotrophic lateral sclerosis (SALS) have a heterogeneous metabolite signature in the cerebrospinal fluid, in some patients being almost identical to controls. However, familial amyotrophic lateral sclerosis (FALS) without superoxide dismutase-1 gene (SOD1) mutation is less heterogeneous than SALS. The metabolome of the cerebrospinal fluid of 17 ALS patients with a SOD1 gene mutation was found to form a separate homogeneous group. Analysis of metabolites revealed that glutamate and glutamine were reduced, in particular in patients with a familial predisposition. There are significant differences in the metabolite profile and composition among patients with FALS, SALS and patients carrying a mutation in the SOD1 gene suggesting that the neurodegenerative process in different subtypes of ALS may be partially dissimilar.

Conclusions/Significance

Patients with a genetic predisposition to amyotrophic lateral sclerosis have a more distinct and homogeneous signature than patients with a sporadic disease.     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PtdIns(3,4,5)P₃ and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia-inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.Key words: filopodia, integrins, migration, cancer     

A secreted BMP antagonist, Cer1, fine tunes the spatial organization of the ureteric bud tree during mouse kidney development

The epithelial ureteric bud is critical for mammalian kidney development as it generates the ureter and the collecting duct system that induces nephrogenesis in dicrete locations in the kidney mesenchyme during its emergence. We show that a secreted Bmp antagonist Cerberus homologue (Cer1) fine tunes the organization of the ureteric tree during organogenesis in the mouse embryo. Both enhanced ureteric expression of Cer1 and Cer1 knock out enlarge kidney size, and these changes are associated with an altered three-dimensional structure of the ureteric tree as revealed by optical projection tomography. Enhanced Cer1 expression changes the ureteric bud branching programme so that more trifid and lateral branches rather than bifid ones develop, as seen in time-lapse organ culture. These changes may be the reasons for the modified spatial arrangement of the ureteric tree in the kidneys of Cer1+ embryos. Cer1 gain of function is associated with moderately elevated expression of Gdnf and Wnt11, which is also induced in the case of Cer1 deficiency, where Bmp4 expression is reduced, indicating the dependence of Bmp expression on Cer1. Cer1 binds at least Bmp2/4 and antagonizes Bmp signalling in cell culture. In line with this, supplementation of Bmp4 restored the ureteric bud tip number, which was reduced by Cer1+ to bring it closer to the normal, consistent with models suggesting that Bmp signalling inhibits ureteric bud development. Genetic reduction of Wnt11 inhibited the Cer1-stimulated kidney development, but Cer1 did not influence Wnt11 signalling in cell culture, although it did inhibit the Wnt3a-induced canonical Top Flash reporter to some extent. We conclude that Cer1 fine tunes the spatial organization of the ureteric tree by coordinating the activities of the growth-promoting ureteric bud signals Gndf and Wnt11 via Bmp-mediated antagonism and to some degree via the canonical Wnt signalling involved in branching.     

hHGF overexpression in myoblast sheets enhances their angiogenic potential in rat chronic heart failure

After severe myocardial infarction (MI), heart failure results from ischemia, fibrosis, and remodeling. A promising therapy to enhance cardiac function and induce therapeutic angiogenesis via a paracrine mechanism in MI is myoblast sheet transplantation. We hypothesized that in a rat model of MI-induced chronic heart failure, this therapy could be further improved by overexpression of the antiapoptotic, antifibrotic, and proangiogenic hepatocyte growth factor (HGF) in the myoblast sheets. We studied the ability of wild type (L6-WT) and human HGF-expressing (L6-HGF) L6 myoblast sheet-derived paracrine factors to stimulate cardiomyocyte, endothelial cell, or smooth muscle cell migration in culture. Further, we studied the autocrine effect of hHGF-expression on myoblast gene expression profiles by use of microarray analysis. We induced MI in Wistar rats by left anterior descending coronary artery (LAD) ligation and allowed heart failure to develop for 4 weeks. Thereafter, we administered L6-WT (n = 15) or L6-HGF (n = 16) myoblast sheet therapy. Control rats (n = 13) underwent LAD ligation and rethoracotomy without therapy, and five rats underwent a sham operation in both surgeries. We evaluated cardiac function with echocardiography at 2 and 4 weeks after therapy, and analyzed cardiac angiogenesis and left ventricular architecture from histological sections at 4 weeks. Paracrine mediators from L6-HGF myoblast sheets effectively induced migration of cardiac endothelial and smooth muscle cells but not cardiomyocytes. Microarray data revealed that hHGF-expression modulated myoblast gene expression. In vivo, L6-HGF sheet therapy effectively stimulated angiogenesis in the infarcted and non-infarcted areas. Both L6-WT and L6-HGF therapies enhanced cardiac function and inhibited remodeling in a similar fashion. In conclusion, L6-HGF therapy effectively induced angiogenesis in the chronically failing heart. Cardiac function, however, was not further enhanced by hHGF expression.     

Increased expression of bradykinin type-1 receptors in endothelium of intramyocardial coronary vessels in human failing hearts

In experimental animals, bradykinin type-1 receptors (BK-1Rs) are induced during inflammation and ischemia, and, by exerting either cardioprotective or cardiotoxic effects, they may contribute to the pathogenesis of heart failure. Nothing is known about the expression of BK-1Rs in human heart failure. Human heart tissue was obtained from excised hearts of patients undergoing cardiac transplantation (n = 13), due to idiopathic dilated cardiomyopathy (IDC; n = 7) or to coronary heart disease (CHD; n = 6), and from normal hearts (n = 6). The expression of BK-1Rs was analyzed by means of competitive RT-PCR, Western blot analysis, and immunohistochemistry. Expression of BK-1R mRNA was increased in both IDC (2.8-fold) and CHD (2.1-fold) hearts compared with normal hearts. The observed changes were verified at the protein level. Expression of BK-1Rs in failing hearts localized to the endothelium of intramyocardial coronary vessels and correlated with an increased expression of TNF-alpha in the vessel wall. Treatment of human coronary artery endothelial cells with TNF-alpha increases their BK-1R expression. These novel results show that BK-1Rs are induced in the endothelium of intramyocardial coronary vessels in failing human hearts and so may participate in the pathogenesis of heart failure.     

Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2

2-Enoyl-CoA hydratase 2 is the middle part of the mammalian peroxisomal multifunctional enzyme type 2 (MFE-2), which is known to be important in the beta-oxidation of very-long-chain and alpha-methyl-branched fatty acids as well as in the synthesis of bile acids. Here, we present the crystal structure of the hydratase 2 from the human MFE-2 to 3A resolution. The three-dimensional structure resembles the recently solved crystal structure of hydratase 2 from the yeast, Candida tropicalis, MFE-2 having a two-domain subunit structure with a C-domain complete hot-dog fold housing the active site, and an N-domain incomplete hot-dog fold housing the cavity for the aliphatic acyl part of the substrate molecule. The ability of human hydratase 2 to utilize such bulky compounds which are not physiological substrates for the fungal ortholog, e.g. CoA esters of C26 fatty acids, pristanic acid and di/trihydroxycholestanoic acids, is explained by a large hydrophobic cavity formed upon the movements of the extremely mobile loops I-III in the N-domain. In the unliganded form of human hydratase 2, however, the loop I blocks the entrance of fatty enoyl-CoAs with chain-length >C8. Therefore, we expect that upon binding of substrates bulkier than C8, the loop I gives way, contemporaneously causing a secondary effect in the CoA-binding pocket and/or active site required for efficient hydration reaction. This structural feature would explain the inactivity of human hydratase 2 towards short-chain substrates. The solved structure is also used as a tool for analyzing the various inactivating mutations, identified among others in MFE-2-deficient patients. Since hydratase 2 is the last functional unit of mammalian MFE-2 whose structure has been solved, the organization of the functional units in the biologically active full-length enzyme is also discussed.     

The role of stochastic and modal gating of cardiac L-type Ca2+ channels on early after-depolarizations

Certain signaling events that promote L-type Ca2+ channel (LCC) phosphorylation, such as beta-adrenergic stimulation or an increased expression of Ca(2+)/calmodulin-dependent protein kinase II, promote mode 2 gating of LCCs. Experimental data suggest the hypothesis that these events increase the likelihood of early after-depolarizations (EADs). We test this hypothesis using an ionic model of the canine ventricular myocyte incorporating stochastic gating of LCCs and ryanodine-sensitive calcium release channels. The model is extended to describe myocyte responses to the beta-adrenergic agonist isoproterenol. Results demonstrate that in the presence of isoproterenol the random opening of a small number of LCCs gating in mode 2 during the plateau phase of the action potential (AP) can trigger EADs. EADs occur randomly, where the likelihood of these events increases as a function of the fraction of LCCs gating in mode 2. Fluctuations of the L-type Ca2+ current during the AP plateau lead to variability in AP duration. Consequently, prolonged APs are occasionally observed and exhibit an increased likelihood of EAD formation. These results suggest a novel stochastic mechanism, whereby phosphorylation-induced changes in LCC gating properties contribute to EAD generation.     

Transient increase of ATP as a response to temperature up-shift in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces the heat shock response to a temperature up-shift which is connected to the synthesis of a characteristic set of proteins, including ATP dependent chaperones and proteases. Therefore the balance of the nucleotide pool is important for the adaptation and continuous function of the cell. Whereas it has been observed in eukaryotic cells, that the ATP level immediately decreased after the temperature shift, no data are available for E. coli about the adenosine nucleotide levels during the narrow time range of minutes after a temperature up-shift.     

Topical superantigen exposure induces epidermal accumulation of CD8+ T cells, a mixed Th1/Th2-type dermatitis and vigorous production of IgE antibodies in the murine model of atopic dermatitis

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVbeta8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-gamma protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.