Repeated application of Modafinil and Levodopa reveals a drug-independent precise timing of spatial working memory modulation.

Cognition enhancing drugs often target the dopaminergic system, which is involved in learning and memory, including working memory that in turn involves mainly the prefrontal cortex and the hippocampus. In most animal models for modulations of working memory animals are pre-trained to a certain criterion and treated then acutely to test drugs effects on working memory. Thus, little is known regarding subchronic or chronic application of cognition enhancing drugs and working memory performance. Therefore we trained male rats over six days in a rewarded alternation test in a T-maze. Rats received daily injections of either modafinil or Levodopa (L-Dopa) at a lower and a higher dose 30min before training. Levodopa but not modafinil increased working memory performance during early training significantly at day 3 when compared to vehicle controls. Both drugs induced dose dependent differences in working memory with significantly better performance at low doses compared to high doses for modafinil, in contrast to L-Dopa where high dose treated rats performed better than low dose rats. Strikingly, these effects appeared only at day 3 for both drugs, followed by a decline in behavioral performance. Thus, a critical drug independent time window for dopaminergic effects upon working memory could be revealed. Evaluating the underlying mechanisms contributes to the understanding of temporal effects of dopamine on working memory performance.

Proteomic analysis of the TerC interactome: Novel links to tellurite resistance and pathogenicity.

The tellurite resistance gene operon (ter) is widely spread among bacterial species, particularly pathogenic species. The ter operon has been implicated in tellurite resistance, phage inhibition, colicine resistance, and pathogenicity. The TerC protein represents one of the key proteins in tellurite resistance and shows no significant homology to any protein of known function. So far, there is no experimental evidence for TerC interaction partners. In this study, proteomic-based methods, including blue native electrophoresis and co-immunoprecipitation combined with LC-MS/MS, have been used to identify TerC interaction partners and thus providing indirect evidence for tentative functions of TerC in Escherichia coli. An interactome has been constructed and robust physical interaction of integral membrane protein TerC with TerB, DctA, PspA, HslU, and RplK has been shown. The TerC-TerB complex appears to act as a central unit that may link different functional modules with biochemical activities of C4-dicarboxylate transport, inner membrane stress response (phage shock protein regulatory complex), ATPase/chaperone activity, and proteosynthesis. In previous reports, it was hypothesized that a transmembrane unit formed by TerC protein may interact with the TerD family, but herein neither TerD nor TerE proteins were identified as TerC complex components. We propose that TerD/TerE participates in tellurite resistance through TerC-independent action.

Zinc deficiency induces enhanced depression-like behaviour and altered limbic activation reversed by antidepressant treatment in mice.

A relationship between zinc (Zn)-deficiency and mood disorders has been suspected. Here we examined for the first time whether experimentally-induced Zn-deficiency in mice would alter depression- and anxiety-related behaviour assessed in established tests and whether these alterations would be sensitive to antidepressant treatment. Mice receiving a Zn-deficient diet (40% of daily requirement) had similar homecage and open field activity compared to normally fed mice, but displayed enhanced depression-like behaviour in both the forced swim and tail suspension tests which was reversed by chronic desipramine treatment. An anxiogenic effect of Zn-deficiency prevented by chronic desipramine and Hypericum perforatum treatment was observed in the novelty suppressed feeding test, but not in other anxiety tests performed. Zn-deficient mice showed exaggerated stress-evoked immediate-early gene expression in the amygdala which was normalised following DMI treatment. Taken together these data support the link between low Zn levels and depression-like behaviour and suggest experimentally-induced Zn deficiency as a putative model of depression in mice.

Hippocampal levels of gamma-enolase, C-1-tetrahydrofolate synthase and serotransferrin fluctuate over the estrous cycle in the rat.

Hippocampal functions vary across the estrous cycle but metabolic changes at the protein level have not been systematically studied so far. It was therefore the aim of the study to screen expression of metabolic proteins mainly represented by metabolic enzymes in the hippocampus over the estrous cycle and in males. Female and male OFA Sprague-Dawley rats were used and female estrous phases were determined by vaginal smears, according to which females were separated into groups of proestrous, estrous, early and late metestrous and diestrous. Proteins were extracted from hippocampal tissue and separated on two-dimensional gel electrophoresis followed by identification with mass spectrometry methods (MALDI-TOF-TOF and nano-LC-ESI-MS/MS). Comparative analysis of protein levels was carried out by quantifying protein spot volumes by means of specific software. Levels of one expression form of gamma-enolase were different between diestrous and early metestrous; C-1-tetrahydrofolate synthase levels were elevated in proestrous as compared with estrous and serotransferrin levels were increased in diestrous as compared with proestrous, estrous, metestrous and in males. The outcome of estrous cycle- and gender-dependent protein fluctuations is relevant for the interpretation of previous and future work as well as for the design of further studies at the protein level in the hippocampus.

Protein expression of BACE1, BACE2 and APP in Down syndrome brains.

Down syndrome (DS) is the most common human chromosomal abnormality caused by an extra copy of chromosome 21. The phenotype of DS is thought to result from overexpression of a gene or genes located on the triplicated chromosome or chromosome region. Several reports have shown that the neuropathology of DS comprises developmental abnormalities and Alzheimer-like lesions such as senile plaques. A key component of senile plaques is amyloid beta-peptide which is generated from the amyloid precursor protein (APP) by sequential action of beta-secretases (BACE1 and BACE2) and gamma-secretase. While BACE1 maps to chromosome 11, APP and BACE2 are located on chromosome 21. To challenge the gene dosage effect and gain insight into the expressional relation between beta-secretases and APP in DS brain, we evaluated protein expression levels of BACE1, BACE2 and APP in fetal and adult DS brain compared to controls. In fetal brain, protein expression levels of BACE2 and APP were comparable between DS and controls. BACE1 was increased, but did not reach statistical significance. In adult brain, BACE1 and BACE2 were comparable between DS and controls, but APP was significantly increased. We conclude that APP overexpression seems to be absent during the development of DS brain up to 18-19 weeks of gestational age. However, its overexpression in adult DS brain could lead to disturbance of normal function of APP contributing to neurodegeneration. Comparable expression of BACE1 and BACE2 speaks against the hypothesis that increased beta-secretase results in (or even underlies) increased production of amyloidogenic A beta fragments. Furthermore, current data indicate that the DS phenotype cannot be fully explained by simple gene dosage effect.

Identification and characterisation of arsenite (+3 Oxidation State) methyltransferase (AS3MT) in mouse neuroblastoma cell line N1E-115.

Handling and detoxification of metals by enzymes is a major issue that is not in the focus of current biomedical research concepts. The finding of the presence of arsenic (+3 Oxidation State) methyltransferase (AS3MT) in neuroblastoma cells NE-115 as a high abundance protein made us investigate primary structure of AS3MT reflecting an example of metal-handling in eucaryotes. Proteins extracted from NE-115 cells were run on 2-DE followed by two different mass spectrometrical methods. High sequence coverage was obtained by multiple protease digestion and a sequence conflict was solved at arginine 335. These findings are important when future studies on this enzyme are designed at the protein level and in particular, when antibodies against this protein will be generated.

Strain-dependent effects of cognitive enhancers in the mouse.

A series of cognitive enhancers (CEs) have been reported to increase spatial memory in rodents, information on behavioral effects, however, is limited. The aim of the study was therefore to examine the behavioral effects of three CEs in two well-documented inbred mouse strains. C57BL/6J and DBA/2 mice were administered intraperitonial. D-cycloserine (DCS; NMDA receptor agonist), 1-(4-Amino-5-chloro-2-methoxyphenyl)-3-[1-butyl-4-piperidinyl]-1-propanone hydrochloride (RS67333; 5HT4-receptor agonist), and (R)-4-{[2-(1-methyl-2-pyrrolidinyl)ethyl]thio}phenol hydrochloride (SIB-1553A; beta-4-nicotinic receptor agonist) and tested in the open field (OF), elevated plus maze (EPM), neurological observational battery and rota-rod. Cognitive performance was tested in the Morris water maze. All compounds modified behavioral performance in the OF, DCS showed an anxiolytic effect in the EPM, and differences in the observational battery were observed i.e. vestibular drop was decreased by SIB-1553A and RS67333 treatment in C57BL/6J and increased with DCS treatment in DBA/2 mice. In the rota rod SIB-1553A improved motor performance. DCS effects on learning and memory was comparable to controls whereas the other compounds impaired performance in the Morris water maze. In conclusion, behavioral testing of CEs in the mouse revealed significant changes that may have to be taken into account for evaluation of CEs, interpretation of cognitive studies and warrant further neurotoxicological studies. Moreover, strain-dependent differences were observed that in turn may confound results obtained from behavioral and cognitive testing.

Mass spectrometrical analysis of the processed metastasis-inducing anterior gradient protein 2 homolog reveals 100% sequence coverage.

Anterior gradient protein 2 homolog is a metastasis-inducing protein in a rat model of rat breast cancer and prognostic for outcome in hormonally treated breast cancer patients. Carrying out protein profiling in several mammalian cells and tissues, we detected this protein (synonym: secreted cement gland protein XAG-2 homolog) that was originally described in toad skin, in human bronchial epithelia. Tissues obtained from biopsies were homogenised and extracted proteins were run on two-dimensional gel electrophoresis. Following in-gel digestion with proteases trypsin, AspN, LysC and chymotrypsin, mass spectrometrical analysis was carried out by MALDI-TOF/TOF. The use of MS following multi-enzyme digestion of the protein resulted into 100% sequence coverage. MS/MS analysis enabled sequencing of 87% of the protein structure. This percentage does not include the signal peptide that was not observed in our protein due to processing. No posttranslational modifications were detectable and no sequence conflicts were observed. Complete analysis, unambiguous identification and characterisation of this biologically important protein could be shown, which is relevant for the definition of a marker protein that has been described so far by immunochemical methods only. Complete analysis is of importance as it forms the basis for all future work on this protein and, moreover, may serve as an analytical tool for further studies.

Start-to-end processing of two-dimensional gel electrophoretic images.

Gel electrophoresis serves as a basic analytical tool in the proteomic studies. However, processing of gel electrophoretic images is still the main bottleneck of data analysis, and there is an increasing need for the fully automated approaches. The proposed start-to-end strategy of analyzing the gel images consists of chemometric tools, which allow their effective preprocessing, automatic warping, and data modeling. The image preprocessing techniques: denoising in the wavelet domain and the penalized asymmetric least squares approach for the background estimation are proposed. Matching of images is based on fuzzy warping of features, extracted from the gel images. For the classification or calibration purpose, multivariate approaches such, as partial least squares (PLS) or kernel-PLS methods are used. Performance of the proposed strategy is demonstrated on the real set of the two-dimensional gel images.

Manifold decrease of sialic acid synthase in fetal Down syndrome brain.

BACKGROUND: Down syndrome (DS, trisomy 21) is the most common genetic cause of mental retardation. A large series of biochemical defects have been observed in fetal and adult DS brain that help in unraveling the molecular mechanisms underlying mental retardation. AIMS: As sialylation of glycoconjugates plays an important role in brain development, this study aimed to look at the sialic acid metabolism by measuring sialic acid synthase (SAS; N-acetylneuraminate synthase) in early second trimester fetal control and DS brain. RESULTS: In this regard, protein profiling was performed by two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass-spectrometry followed by database search and subsequent quantification of spot using specific software. SAS, the enzyme catalyzing synthesis of N-acetyl-neuraminic acid (syn: sialic acid) was represented as a single spot and found to be significantly and manifold reduced (P < 0.01) in cortex of fetuses with DS (control vs. DS, 0.052 +/- 0.025 vs. 0.012 +/- 0.006). CONCLUSION: The intriguing finding of the manifold decrease of SAS in DS fetal cerebral cortex as early as in the second trimester of pregnancy may help to explain the brain deficit observed in DS. Decreased SAS may well lead to altered sialic acid metabolism, required for brain development and, more specifically, for sialylation of key brain proteins, including neuronal cell adhesion molecule and myelin associated glycoprotein.

Autotaxin (NPP-2) in the brain: cell type-specific expression and regulation during development and after neurotrauma.

Autotaxin is a secreted cell motility-stimulating exo-phosphodiesterase with lysophospholipase D activity that generates bioactive lysophosphatidic acid. Lysophosphatidic acid has been implicated in various neural cell functions such as neurite remodeling, demyelination, survival and inhibition of axon growth. Here, we report on the in vivo expression of autotaxin in the brain during development and following neurotrauma. We found that autotaxin is expressed in the proliferating subventricular and choroid plexus epithelium during embryonic development. After birth, autotaxin is mainly found in white matter areas in the central nervous system. In the adult brain, autotaxin is solely expressed in leptomeningeal cells and oligodendrocyte precursor cells. Following neurotrauma, autotaxin is strongly up-regulated in reactive astrocytes adjacent to the lesion. The present study revealed the cellular distribution of autotaxin in the developing and lesioned brain and implies a function of autotaxin in oligodendrocyte precursor cells and brain injuries.

Mass spectrometrical analysis of galectin proteins in primary rat cerebellar astrocytes.

Galectins are a family of animal lectins with specificity for beta-galactosides and are involved in a host of cellular activities, ranging from development to cancer. The molecules are expressed by neural and non-neural cells intracellularly as well as extracellularly. Using two-dimensional gel electrophoresis coupled to tandem mass spectrometry, the present work aimed to identify and characterize galectins in primary rat cerebellar astrocytes. The protein-chemical method identified nine spots representing two members of the galectin family, namely galectin-1 and galectin-3. These findings suggest that high abundant expression of galectin in astrocytes is limited to the two abundant galectin family members. As these family members are linked to human astrocytic tumors, their reliable detection in astrocytes by proteomic techniques would enable us to further understand their role in neural development, injury, and regeneration in general and astrocytoma in particular.

Nectin-like molecule 1 is a high abundance protein in cerebellar neurons.

Nectins and Nectin-like molecules belong to the Ca-independent immunoglobulin superfamily of cell adhesion molecules and are mandatory for various cellular functions such as morphogenesis, differentiation and proliferation. Among them, Nectin-like molecule 1 (Necl-1) is unique for its exclusive expression in the brain where it is localized at the contact sites among axon terminals and glia cell processes, cooperatively forming synapses. We hereby aimed to unambiguously characterize Necl-1 at the protein level in rat brain. Rat cerebellar neurons were lysed, proteins extracted and run on two-dimensional gel electrophoresis with subsequent in-gel digestion and mass spectrometrical (MS/MS) analysis of protein spots. One spot at pI 5.96 with an observed molecular weight of 26 kDa was identified as Nectin-like molecule 1. MS/MS analyses of three matching peptides warranted unambiguous identification for the first time. Additionally, we verified the result by immunoblotting and detected two bands at about 48 kDa and 60 kDa. The proposed roles of Necl-1 in cerebellar morphogenesis as well as plasticity of synapses challenge further research on its function in more detail and we hereby provide a fair analytical tool for the unequivocal determination of Necl-1, independent of antibody availability and specificity.

Tuberin activates the proapoptotic molecule BAD.

TSC1, encoding hamartin, and TSC2, encoding tuberin, are tumor suppressor genes responsible for the autosomal dominantly inherited disease tuberous sclerosis (TSC). TSC affects approximately 1 in 6000 individuals and is characterized by the development of tumors, named hamartomas, in different organs. Hamartin and tuberin form a complex, of which tuberin is assumed to be the functional component. The TSC proteins have been implicated in the control of cell cycle and cell size. In addition to enhanced growth, reduced death rates can lead to tumor development. Therefore, defects in the apoptosis-inducing pathways contribute to neoplastic cell expansion. Here, we show that tuberin triggers apoptosis, accompanied by downregulation of p70S6K activity and of phosphorylation of BAD on residue Ser136, and by upregulation of the interaction of BAD/BCL-2 and BAD/BCL-XL. AKT phosphorylation negatively regulates tuberin's potential to trigger apoptosis. Experiments with BAD-/- cells demonstrate BAD to be a mediator of tuberin's effects on the regulation of apoptosis. Tuberin interferes with insulin-like growth factor-1-induced BAD Ser136 phosphorylation and cell survival. Our work proposes a model in which tuberin-mediated inhibition of p70S6K activates BAD to heterodimerize with BCL-2 and BCL-XL to promote apoptosis. A mutation of TSC2--as it occurs in TSC patients--attenuates this proapoptotic potential, underscoring the relevance of our findings for human pathophysiology.

Mass-spectrometrical analysis of proteins encoded on chromosome 21 in human fetal brain.

Overexpression of chromosome 21 genes is directly or indirectly responsible for the Down syndrome phenotype. In order to analyse chromosome 21 gene products (Chr21Ps), we extracted proteins from fetal human brain cortex and applied an ultracentrifugal and chromatographic prefractionation principle followed by two-dimensional gel electrophoresis (2-DE) and mass-spectrometrical analysis using high-throughput automated MALDI-TOF/TOF. Nine Chr21Ps were identified: pyridoxal kinase; superoxide dismutase [Cu/Zn] 1; carbonyl reductase 1; ES1 protein homolog, mitochondrial [Precursor]; cystathionine-beta-synthetase; T-complex protein 1, theta subunit; cystatin B; 6-phosphofructokinase; glycinamide ribonucleotide synthetase. Mass-spectrometric characterisation of Chr21Ps following separation in 2-DE gels is a useful tool for the analysis of these structures in brain, independent of antibody availability and specificity.

Aberrant expression of cytoskeleton proteins in hippocampus from patients with mesial temporal lobe epilepsy.

Mesial temporal lobe epilepsy (MTLE), the most common form of epilepsy, is characterised by cytoarchitectural abnormalities including neuronal cell loss and reactive gliosis in hippocampus. Determination of aberrant cytoskeleton protein expression by proteomics techniques may help to understand pathomechanism that is still elusive. We searched for differential expression of hippocampal proteins by an analytical method based on two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry unambiguously identifying 77 proteins analysed in eight control and eight MTLE hippocampi. Proteins were quantified and we observed 18 proteins that were altered in MTLE. Cytoskeleton proteins tubulin alpha-1 chain, beta-tubulin, profilin II, neuronal tropomodulin were significantly reduced and one actin spot was missing, whereas ezrin and vinculin were significantly increased in MTLE. Proteins of several classes as e.g. antioxidant proteins (peroxiredoxins 3 and 6), chaperons (T-complex protein 1-alpha, stress-induced-phosphoprotein 1), signaling protein MAP kinase kinase 1, synaptosomal proteins (synaptotagmin I, alpha-synuclein), NAD-dependent deacetylase sirtuin-2 and 26S protease regulatory subunit 7 protein, neuronal-specific septin 3 were altered in MTLE. Taken together, the findings may represent or lead to cytoskeletal impairment; aberrant antioxidant proteins, chaperons, MAP kinase kinase 1 and NAD-dependent deacetylase sirtuin-2 may have been involved in pathogenetic mechanisms and altered synaptosomal protein expression possibly reflects synaptic impairment in MTLE.

Brain G protein-dependent signaling pathways in Down syndrome and Alzheimer's disease.

Premature aging and neuropathological features of Alzheimer's disease (AD) are commonly observed in Down syndrome (DS). Based on previous findings in a DS mouse model, the function of signaling pathways associated with adenylyl cyclase (AC) and phospholipase C (PLC) was assessed in cerebral cortex and cerebellum of age-matched adults with DS, AD, and controls. Basal production of cAMP was reduced in DS but not in AD cortex, and in both, DS and AD cerebellum. Responses to GTPgammaS, noradrenaline, SKF 38393 and forskolin were more depressed in DS than in AD cortex and cerebellum. Although no differences in PLC activity among control, DS and AD cortex were observed under basal and GTPgammaS- or Ca-stimulated conditions, the response of DS cortex to serotonergic and cholinergic stimulation was depressed, and that of AD was only impaired at cholinergic stimulation. No differences were documented in cerebellum. Our results demonstrate that PLC and AC were severely disturbed in the aged DS and AD brains, but the alterations in DS were more severe, and differed to some extent from those observed in AD.

Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.

Strain-dependent regulation of neurotransmission and actin-remodelling proteins in the mouse hippocampus.

Individual mouse strains differ significantly in terms of behaviour, cognitive function and long-term potentiation. Hippocampal gene expression profiling of eight different mouse strains points towards strain-specific regulation of genes involved in neuronal information storage. Protein expression with regard to strain- dependent expression of structures related to neuronal information storage has not been investigated yet. Herein, a proteomic approach based on two-dimensional gel electrophoresis coupled with mass spectrometry (MALDI-TOF/TOF) has been chosen to address this question by determining strain-dependent expression of proteins involved in neurotransmission and activity-induced actin remodelling in hippocampal tissue of five mouse strains. Of 31 spots representing 16 different gene products analysed and quantified, N-ethylmaleimide-sensitive fusion protein, N-ethylmaleimide-sensitive factor attachment protein-alpha, actin-like protein 3, profilin and cofilin were expressed in a strain-dependent manner. By treating protein expression as a phenotype, we have shown significant genetic variation in brain protein expression. Further experiments in this direction may provide an indication of the genetic elements that contribute to the phenotypic differences between the selected strains through the expressional level of the translated protein. In view of this, we propose that proteomic analysis enabling to concomitantly survey the expression of a large number of proteins could serve as a valuable tool for genetic and physiological studies of central nervous system function.

The neuronal differentiation process involves a series of antioxidant proteins.

Involvement of individual antioxidant proteins (AOXP) and antioxidants in the differentiation process has been already reported. A systematic search strategy for detecting differentially regulated AOXP in neuronal differentiation, however, has not been published so far. The aim of this study was to provide an analytical tool identifying AOXP and to generate a differentiation-related AOXP expressional pattern. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments: We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical (MALDI-TOF-TOF) identification of proteins to generate a map of AOXP. 16 AOXP were unambiguously determined in both cell lines; catalase, thioredoxin domain-containing protein 4 and hypothetical glutaredoxin/glutathione S-transferase C terminus-containing protein were detectable in the undifferentiated cells only. Five AOXP were observed in both, undifferentiated and differentiated cells and thioredoxin, thioredoxin-like protein p19, thioredoxin reductase 1, superoxide dismutases (Mn and Cu-Zn), glutathione synthetase, glutathione S-transferase P1 and Mu1 were detected in differentiated cells exclusively. Herein a differential expressional pattern is presented that reveals so far unpublished antioxidant principles involved in neuronal differentiation by a protein chemical approach, unambiguously identifying AOXP. This finding not only shows concomitant determination of AOXP but also serves as an analytical tool and forms the basis for design of future studies addressing AOXP and differentiation per se.