Data in support of metabolic reprogramming in transformed mouse cortical astrocytes: A proteomic study.

2D-DIGE analysis coupled with mass spectrometry is a global, without a priori, comparative proteomic approach particularly suited to identify and quantify enzymes isoforms and structural proteins, thus making it an efficient tool for the characterization of the changes in cell phenotypes that occur in physiological and pathological conditions. In this data article in support of the research article entitled "Metabolic reprogramming in transformed mouse cortical astrocytes: a proteomic study" [1] we illustrate the changes in protein profile that occur during the metabolic reprogramming undergone by cultured mouse astrocytes in a model of in-vitro cancerous transformation [2].

Metabolic reprogramming in transformed mouse cortical astrocytes: A proteomic study.

Metabolic reprogramming is thought to play a key role in sustaining the survival and proliferation of cancer cells. These changes facilitate for example the uptake and release of nutrients required for nucleotide, protein and lipid synthesis necessary for macromolecule assembly and tumor growth. We applied a 2D-DIGE (two-dimensional differential in-gel electrophoresis) quantitative proteomic analysis to characterize the proteomes of mouse astrocytes that underwent in vitro cancerous transformation, and of their normal counterparts. Metabolic reprogramming effects on enzymatic and structural protein expression as well as associated metabolites abundance were quantified. Using enzymatic activity measurements and zymography, we documented and confirmed several changes in abundance and activity of various isoenzymes likely to participate in metabolic reprogramming. We found that after transformation, the cells increase their expression of glycolytic enzymes, thus augmenting their ability to use aerobic glycolysis (Warburg effect). An increased capacity to dispose of reducing equivalents through lactate production was also documented. Major effects on carbohydrates, amino acids and nucleotides metabolic enzymes were also observed. Conversely, the transformed cells reduced their enzymatic capacity for reactions of tricarboxylic acid oxidation, for neurotransmitter (glutamate) metabolism, for oxidative stress defense and their expression of astroglial markers. BIOLOGICAL SIGNIFICANCE: The use of a global approach based on a 2D DIGE analysis allows obtaining a comprehensive view of the metabolic reprogramming undergone by astrocytes upon cancerous transformation. Indeed, except for a few enzymes such as pyruvate carboxylase and glutaminase that were not detected in our initial analysis, pertinent information on the abundance of most enzymes belonging to pathways relevant to metabolic reprogramming was directly obtained. In this in vitro model, transformation causes major losses of astrocyte-specific proteins and functions and the acquisition of metabolic adaptations that favor intermediate metabolites production for increased macromolecule biosynthesis. Thus our approach appears to be readily applicable for the investigation of changes in protein abundance that determine various transformed cell phenotypes. It could similarly be applied to the evaluation of the effects of treatments aimed at correcting the consequences of cell transformation.

Comprehensive plasma profiling for the characterization of graft-versus-host disease biomarkers.

Acute graft-versus-host disease (aGVHD) remains a life-threatening complication of hematopoietic stem cell transplantation (HSCT) therefore limiting its application. To optimize the management of aGVHD and reduce therapy-related toxicity, early specific markers are needed. The main objective of this study was to uncover diagnostic biomarkers by comparing plasma protein profiles of patients at the time of acute GVHD diagnosis with those of patients undergoing HSCT without aGVHD. Additional analysis of samples taken 15 days before aGVHD diagnosis was also performed to evaluate the potential of our newly discovered biomarkers for early diagnosis. To get complementary information from plasma samples, we used three different proteomic approaches, namely 2D-DIGE, SELDI-TOF-MS and 2D-LC-MS(E). We identified and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased inflammation response and disturbance in the coagulation cascade. The variation of these proteins was already observed 15 days before GVHD diagnosis, suggesting the potential early detection of the disease before symptoms appearance. Finally, logistic regression analysis determined a composite biomarker panel comprising fibrinogen, fragment of fibrinogen beta chain, SAA, prothrombin fragments, apolipoprotein A1 and hepcidin that optimally discriminated patients with and without GVHD. The area under the receiver operating characteristic curve distinguishing these 2 groups was 0.95.

Inactivation of genes coding for mitochondrial Nd7 and Nd9 complex I subunits in Chlamydomonas reinhardtii. Impact of complex I loss on respiration and energetic metabolism.

In Chlamydomonas, unlike in flowering plants, genes coding for Nd7 (NAD7/49 kDa) and Nd9 (NAD9/30 kDa) core subunits of mitochondrial respiratory-chain complex I are nucleus-encoded. Both genes possess all the features that facilitate their expression and proper import of the polypeptides in mitochondria. By inactivating their expression by RNA interference or insertional mutagenesis, we show that both subunits are required for complex I assembly and activity. Inactivation of complex I impairs the cell growth rate, reduces the respiratory rate, leads to lower intracellular ROS production and lower expression of ROS scavenging enzymes, and is associated to a diminished capacity to concentrate CO2 without compromising photosynthetic capacity.

Effects of eccentrically and concentrically biased training on mouse muscle phenotype.

INTRODUCTION: The molecular adaptations specifically induced by different muscle contraction types have only been partially elucidated. We previously demonstrated that eccentric contractions in human quadriceps elicited proteome modifications that suggest a muscle fiber typology adaptation. We address this question in a more systematic way by examining here the effects of different running modes on the mouse muscle proteome and the muscle fiber typology. METHODS: Male adult mice (C57BL6) were randomly divided into downhill running (DHR) (quadricipital eccentrically biased contractions), uphill running (UHR) (quadricipital concentrically biased contractions), and untrained control (CONT) groups. Running groups performed five training sessions on an inclined treadmill for 75 to 135 min · d(-1), and the quadriceps muscles were dissected 96 h after the last session. Muscle protein extracts of DHR and UHR groups (n = 4/group) were subjected to a two-dimensional difference in gel electrophoresis (2D-DIGE) analysis coupled with mass spectrometry. The assessment of fiber type, size, and number was performed on the rectus femoris of the three groups (n = 6/group) using myosin heavy chain immunohistochemistry. RESULTS: In the proteomic analysis, eight spots identified as the fast myosin heavy chain isoforms exhibited a lower abundance in DHR compared with UHR (P < 0.05, t-test). In contrast, adenosine triphosphate (ATP) synthase subunit α and tubulin ß were more expressed in DHR (P < 0.05). A significant higher proportion of Type I and IIa fibers was found for DHR compared with UHR or CONT groups (P < 0.05, one-way ANOVA). CONCLUSIONS: Our data suggest that the eccentrically biased contractions in mice induced specific adaptations in protein expression and muscle fiber composition, which may reflect a more oxidative muscle phenotype. The differences in stress placed on the muscle between both trainings may be responsible for some unique adaptations resulting from the eccentrically biased training.

Mesenchymal stem cells and neural crest stem cells from adult bone marrow: characterization of their surprising similarities and differences.

The generation of neuronal cells from stem cells obtained from adult bone marrow is of significant clinical interest in order to design new cell therapy protocols for several neurological disorders. The recent identification in adult bone marrow of stem cells derived from the neural crest stem cells (NCSC) might explain the neuronal phenotypic plasticity shown by bone marrow cells. However, little information is available about the nature of these cells compared to mesenchymal stem cells (MSC), including their similarities and differences. In this paper, using transcriptomic as well as proteomic technologies, we compared NCSC to MSC and stromal nestin-positive cells, all of them isolated from adult bone marrow. We demonstrated that the nestin-positive cell population, which was the first to be described as able to differentiate into functional neurons, was a mixed population of NCSC and MSC. More interestingly, we demonstrated that MSC shared with NCSC the same ability to truly differentiate into Tuj1-positive cells when co-cultivated with paraformaldehyde-fixed cerebellar granule neurons. Altogether, those results suggest that both NCSC and MSC can be considered as important tools for cellular therapies in order to replace neurons in various neurological diseases.

Human muscle proteome modifications after acute or repeated eccentric exercises.

INTRODUCTION: Delayed-onset muscle soreness (DOMS), a condition triggered by eccentric exercise, affects muscle cells at a biochemical level in a poorly understood fashion. The objective of the present study was to examine human muscle proteome modifications induced by strenuous eccentric exercises after a specific training aimed to prevent DOMS. METHODS: Biopsy samples of the rectus femoris were obtained from healthy human volunteers in three successive conditions: 1) at rest, 2) 24 h after an injuring exercise protocol consisting of three series of 30 maximal contractions of the quadriceps on an isokinetic dynamometer, and 3) 24 h after a similar exercise bout preceded either by five eccentric training sessions or by no training. RESULTS: Muscle damage was assessed before and 1 d after each maximal eccentric test by comparing three indirect markers: plasma activity of creatine kinase, muscle stiffness, and subjective pain intensity. Compared with the first eccentric test, those markers were reduced after the second test and further reduced if this second test followed the eccentric training, thus confirming the protective effect of such training. Muscle protein extracts were subjected to a two-dimensional difference gel electrophoresis proteomic analysis coupled with matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry protein identification. Surprisingly, we observed that myosin heavy chains decreased after the first eccentric test and were reduced further with other contractile proteins after the second test. Furthermore, the expression of several glycolytic enzymes decreased only after the second test, which was preceded by a specific training. CONCLUSIONS: These findings suggest that the eccentric training resulted in a switch to oxidative metabolism, which may be associated with protection from DOMS.

Proteomics of life at low temperatures: trigger factor is the primary chaperone in the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125.

The proteomes expressed at 4 degrees C and 18 degrees C by the psychrophilic Antarctic bacterium Pseudoalteromonas haloplanktis have been compared using two-dimensional differential in-gel electrophoresis, showing that translation, protein folding, membrane integrity and anti-oxidant activities are upregulated at 4 degrees C. This proteomic analysis revealed that the trigger factor is the main upregulated protein at low temperature. The trigger factor is the first molecular chaperone interacting with virtually all newly synthesized polypeptides on the ribosome and also possesses a peptidyl-prolyl cis-trans isomerase activity. This suggests that protein folding at low temperatures is a rate-limiting step for bacterial growth in cold environments. It is proposed that the psychrophilic trigger factor rescues the chaperone function as both DnaK and GroEL (the major bacterial chaperones but also heat-shock proteins) are downregulated at 4 degrees C. The recombinant psychrophilic trigger factor is a monomer that displays unusually low conformational stability with a Tm value of 33 degrees C, suggesting that the essential chaperone function requires considerable flexibility and dynamics to compensate for the reduction of molecular motions at freezing temperatures. Its chaperone activity is strongly temperature-dependent and requires near-zero temperature to stably bind a model-unfolded polypeptide.

The proapoptotic C16-ceramide-dependent pathway requires the death-promoting factor Btf in colon adenocarcinoma cells.

Ceramides are central molecules in sphingolipid metabolism. They are involved in the regulation of cancer-cell growth, differentiation, senescence and apoptosis. To better understand how these secondary messengers induce their biological effects, adenocarcinoma cells (HCT116) were treated with exogenous long-chain ceramides (C16-ceramide) in order to mimic endogenous sphingolipids. This treatment induced a decrease of cell viability partly due to apoptosis as shown by PARP cleavage and a decrease of pro-caspase 3. Two-dimensional differential in-gel electrophoresis (2D-DIGE) revealed the differential expression of 51 proteins in response to C16-ceramide. These proteins are notably involved in cell proliferation, apoptosis, protein transport and transcriptional regulation. Among them, the cell death-promoting factor Btf was found to be implicated in the apoptotic signal triggered by ceramide. In adenocarcinoma cells, Btf regulates apoptosis related proteins such as Mdm2, p53, BAX and pBcl-2 and thus plays an important role in the ceramide mediated cell death. These findings bring new insight into the proapoptotic ceramide-dependent signaling pathway.

Leukemia inhibitory factor induces an antiapoptotic response in oligodendrocytes through Akt-phosphorylation and up-regulation of 14-3-3.

Leukemia inhibitory factor (LIF) promotes the survival of oligodendrocytes (OLG) both in vitro and in an animal model of multiple sclerosis. Here, we show that LIF protects mature rat OLG cultures selectively against the combined insult of the proinflammatory cytokines interferon-gamma and tumor necrosis factor-alpha, but it does not protect against oxidative stress nor against staurosporine induced apoptosis. We further demonstrate that LIF activates the janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) and the phosphatidylinositol 3 kinase/Akt pathway in mature OLG. We show that LIF protection is independent of suppressors of cytokine signaling and Bcl-2 mRNA expression levels. To gain further insight into the protective mechanism, a quantitative proteomic approach (DIGE) was applied to identify differentially expressed proteins in LIF-treated OLG. Our results indicate that LIF induces a shift in the cellular machinery toward a prosurvival execution program, illustrated by an enhanced expression of isoforms of the antiapoptotic molecule 14-3-3. These data provide further insight into the mechanisms of LIF-mediated protection of mature OLGs.

Regulation of nestin expression by thrombin and cell density in cultures of bone mesenchymal stem cells and radial glial cells.

BACKGROUND: Bone marrow stromal cells and radial glia are two stem cell types with neural phenotypic plasticity. Bone marrow mesenchymal stem cells can differentiate into osteocytes, chondrocytes and adipocytes, but can also differentiate into non-mesenchymal cell, i.e. neural cells in appropriate in vivo and in vitro experimental conditions. Likewise, radial glial cells are the progenitors of many neurons in the developing cortex, but can also generate astrocytes. Both cell types express nestin, an intermediate filament protein which is the hallmark of neural precursors. RESULTS: In this study, we demonstrate that thrombin, a multifunctional serine protease, stimulates the growth of radial glial cells (RG) and mesenchymal stem cells (MSCs) in a dose-dependent manner. In RG, the mitogenic effect of thrombin is correlated with increased expression of nestin but in MSCs, this mitogenic effect is associated with nestin down-regulation. Both cell types express the PAR-1 type receptor for Thrombin and the effect of Thrombin on both cell types can be mimicked by its analogue TRAP-6 activating specifically this receptor subtype or by serum which contains various amount of thrombin. Moreover, we also demonstrate that serum deprivation-induced expression of nestin in MSCs is inhibited by high cell density (> 50,000 cells/cm2). CONCLUSION: This work shows that thrombin stimulates the growth of both RG and MSCs and that nestin expression by MSCs and RG is regulated in opposite manner by thrombin in vitro. Thrombin effect is thus associated in both cell types with a proliferating, undifferentiated state but in RG this involves the induction of nestin expression, a marker of immaturity for neural progenitors. In MSCs however, nestin expression, as it corresponds to a progression from the mesenchymal "undifferentiated", proliferating phenotype toward acquisition of a neural fate, is inhibited by the mitogenic signal.

Steatosis-induced proteomic changes in liver mitochondria evidenced by two-dimensional differential in-gel electrophoresis.

Steatosis encompasses the accumulation of droplets of fats into hepatocytes. In this work, we performed a comparative analysis of mitochondrial protein patterns found in wild-type and steatosis-affected liver using the novel technique two-dimensional differential in-gel electrophoresis (2D-DIGE). A total of 56 proteins exhibiting significant difference in their abundances were unambiguously identified. Interestingly, major proteins that regulate generation and consumption of the acetyl-CoA pool were dramatically changed during steatosis. Many proteins involved in the response to oxidative stress were also affected.

Identification by two-dimensional electrophoresis of a new adhesin expressed by a low-passaged strain of Mycoplasma bovis.

A significant decrease in adherence rates of Mycoplasma bovis to bovine bronchial epithelial (BBE) cells has been observed after passage of the organism in artificial medium. Analysis of the proteins expressed by M. bovis isolate 2610 by two-dimensional (2-D) electrophoresis demonstrated differences between the cells harvested after the 7th and 116th passage. Three silver-stained prominent spots observed in 2-D electrophoretic separation of protein extracts of the lower-passaged cells were considerably less strongly expressed in the sample from higher-passaged cells. These spots had a molecular mass of approximately 24 kDa and an isoelectric point of about 5. The mass spectrometry analysis of these trypsin-sensitive proteins led to their identification as a unique new member of the Vsps family of membrane-associated proteins. Serum from a mouse immunized with these proteins significantly reduced adherence of M. bovis to BBE cells. This result underlines the function of this new Vsp in adherence of M. bovis to host cells.

Plasticity of cultured mesenchymal stem cells: switch from nestin-positive to excitable neuron-like phenotype.

Bone marrow mesenchymal stem cells (MSCs) can differentiate into several types of mesenchymal cells, including osteocytes, chondrocytes, and adipocytes, but, under appropriate experimental conditions, can also differentiate into nonmesenchymal cells--for instance, neural cells. These observations have raised interest in the possible use of MSCs in cell therapy strategies for various neurological disorders. In the study reported here, we addressed the question of in vitro differentiation of MSCs into functional neurons. First, we demonstrate that when they are co-cultured with cerebellar granule neurons, adult MSCs can express neuronal markers. Two factors are needed for the emergence of neuronal differentiation of the MSCs: the first one is nestin expression by MSCs (nestin is a marker for the responsive character of MSCs to extrinsic signals), and the second one is a direct cell-cell interaction between neural cells and MSCs that allows the integration of these extrinsic signals. Three different approaches suggest that neural phenotypes arise from MSCs by a differentiation rather than a cell fusion process, although this last phenomenon can also coexist. The expression of several genes--including sox, pax, notch, delta, frizzled, and erbB--was analyzed by quantitative reverse transcription polymerase chain reaction (RT-PCR) in order to further characterize the nestin-positive phenotype compared to the nestin-negative one. An overexpression of sox2, sox10, pax6, fzd, erbB2, and erbB4 is found in nestin-positive MSCs. Finally, electrophysiological analyses demonstrate that MSC-derived neuron-like cells can fire single-action potentials and respond to several neurotransmitters such as GABA, glycine, and glutamate. We conclude that nestin-positive MSCs can differentiate in vitro into excitable neuron-like cells.

Nestin-positive mesenchymal stem cells favour the astroglial lineage in neural progenitors and stem cells by releasing active BMP4.

BACKGROUND: Spontaneous repair is limited after CNS injury or degeneration because neurogenesis and axonal regrowth rarely occur in the adult brain. As a result, cell transplantation has raised much interest as potential treatment for patients with CNS lesions. Several types of cells have been considered as candidates for such cell transplantation and replacement therapies. Foetal brain tissue has already been shown to have significant effects in patients with Parkinson's disease. Clinical use of the foetal brain tissue is, however, limited by ethical and technical problems as it requires high numbers of grafted foetal cells and immunosuppression. Alternatively, several reports suggested that mesenchymal stem cells, isolated from adult bone marrow, are multipotent cells and could be used in autograft approach for replacement therapies. RESULTS: In this study, we addressed the question of the possible influence of mesenchymal stem cells on neural stem cell fate. We have previously reported that adult rat mesenchymal stem cells are able to express nestin in defined culture conditions (in the absence of serum and after 25 cell population doublings) and we report here that nestin-positive (but not nestin-negative) mesenchymal stem cells are able to favour the astroglial lineage in neural progenitors and stem cells cultivated from embryonic striatum. The increase of the number of GFAP-positive cells is associated with a significant decrease of the number of Tuj1- and O4-positive cells. Using quantitative RT-PCR, we demonstrate that mesenchymal stem cells express LIF, CNTF, BMP2 and BMP4 mRNAs, four cytokines known to play a role in astroglial fate decision. In this model, BMP4 is responsible for the astroglial stimulation and oligodendroglial inhibition, as 1) this cytokine is present in a biologically-active form only in nestin-positive mesenchymal stem cells conditioned medium and 2) anti-BMP4 antibodies inhibit the nestin-positive mesenchymal stem cells conditioned medium inducing effect on astrogliogenesis. CONCLUSIONS: When thinking carefully about mesenchymal stem cells as candidates for cellular therapy in neurological diseases, their effects on resident neural cell fate have to be considered.