ABSTRACT: BACKGROUND: Human cerebral spinal fluid (CSF) is known to be a rich source of small molecule biomarkers for neurological and neurodegenerative diseases. In 2007, we conducted a comprehensive metabolomic study and performed a detailed literature review on metabolites that could be detected (via metabolomics or other techniques) in CSF. A total of 308 detectable metabolites were identified, of which only 23% were shown to be routinely identifiable or quantifiable with the metabolomics technologies available at that time. The continuing advancement in analytical technologies along with the growing interest in CSF metabolomics has led us to re-visit the human CSF metabolome and to re-assess both its size and the level of coverage than can be achieved with today's technologies. METHODS: We used five analytical platforms, including nuclear magnetic resonance (NMR), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), direct flow injection-mass spectrometry (DFI-MS/MS) and inductively coupled plasmas-mass spectrometry (ICP-MS) to perform quantitative metabolomics on multiple human CSF samples. This experimental work was complemented with an extensive literature review to acquire additional information on reported CSF compounds, their concentrations and their disease associations. RESULTS: NMR, GC-MS and LC-MS methods allowed the identification and quantification of 70 CSF metabolites (as previously reported). DFI-MS/MS allowed the quantification of 78 metabolites (6 acylcarnitines, 13 amino acids, hexose, 42 phosphatidylcholines, 2 lyso-phosphatidylcholines and 14 sphingolipids), while ICP-MS provided quantitative results for 33 metal ions in CSF. Literature analysis led to the identification of 57 more metabolites. In total, 476 compounds have now been confirmed to exist in human CSF. CONCLUSIONS: The use of improved metabolomic and other analytical techniques has led to a 54% increase in the known size of the human CSF metabolome over the past 5 years. Commonly available metabolomic methods, when combined, can now routinely identify and quantify 36% of the 'detectable' human CSF metabolome. Our experimental works measured 78 new metabolites that, as per our knowledge, have not been reported to be present in human CSF. An updated CSF metabolome database containing the complete set of 476 human CSF compounds, their concentrations, related literature references and links to their known disease associations is freely available at the CSF metabolome database.

Abstract Objective: To evaluate the use of metabolomics for the first-trimester detection of maternal metabolic dysfunction in and prediction of subsequent development of early-onset preeclampsia. Study Design: This was a case-control study of maternal plasma samples collected at 11-13 weeks' gestation from 30 women that subsequently developed preeclampsia requiring delivery before 34 weeks and 60 unaffected controls. Nuclear magnetic Resonance (NMR) spectroscopy was used to identify and quantify metabolomic changes in cases versus controls. Both genetic computing and standard statistical analyses were performed to predict the development of preeclampsia from the metabolite concentrations alone as well as the combination of metabolite concentrations with maternal characteristics and first trimester uterine artery Doppler pulsatility index (PI). Results: Significant differences between cases and controls were found for 20 metabolites. A combination of four of these metabolites (citrate, glycerol, hydroxy-isovalerate and methionine) appeared highly predictive of preeclampsia with an estimated detection rate of 75.9%, at a false positive rate of 4.9%. The predictive performance was improved by the addition of uterine artery Doppler PI and fetal crown rump length (CRL) and with an estimated detection rate of 82.6%, at a false positive rate of 1.6%. Conclusion: A profound change in the first-trimester metabolite profile was noted in women that subsequently developed early-onset preeclampsia. Preliminary algorithms appeared highly sensitive for first-trimester prediction of preeclampsia.

Systemic administration of the Group II metabotropic glutamate (mGlu) receptor agonist, LY379268 (LY37), dose-dependently suppresses rapid eye movement sleep (REM) whereas systemic administration of the mGlu II receptor antagonist, LY341495 (LY34), increases arousal. Group II mGlu receptors are highly expressed in the amygdala, a brain region involved in the regulation of sleep and arousal. To determine whether the amygdala is involved in mediating the effects of Group II mGlu agents on sleep, we microinjected LY37 and LY34 into the basal amygdala (BA) and the central nucleus of the amygdala (CNA) and recorded sleep and wakefulness. Wistar rats were implanted with electrodes for recording sleep and with bilateral cannulae aimed into BA for drug administration. Different groups of rats received bilateral microinjections of LY37 into BA at two dosage ranges (3.2mM, 5.3mM or 10.7mM or 0.1nM, 2.0nM or 10.0nM) or one dosage range of LY34 (1.0nM, 30.0nM or 60.0nM). Microinjections into CNA were conducted at one dosage range for LY37 (0.1nM, 2.0nM or 10.0nM) and for LY34 (1.0nM, 30.0nM or 60.0nM). All drugs or vehicle alone were administered in a counterbalanced order at 5-day intervals. Following microinjection, sleep was recorded for 20h. Microinjection of LY37 into BA at both nM and mM concentrations significantly decreased REM without significantly altering NREM, total sleep or wakefulness. The high dosage of LY34 in BA significantly suppressed NREM and total sleep. Microinjections of LY37 or LY34 into CNA had no significant impact on sleep. We suggest that Group II mGlu receptors may influence specific cells in BA that control descending output (via the CNA or bed nucleus of the stria terminalis) that in turn regulates pontine REM generator regions.

Nano alumina, one of the most important nanomaterials, is widely used in diverse areas. It was reported that nano alumina could cross the blood brain barrier to enter the brain. Considering aluminum accumulation in brain is closely related to many neural diseases. We studied the neural toxicity of four nano gamma-alumina samples by using neural stem cells (NSCs) C17.2 as a model. We find that the toxicity of nano gamma-alumina is pretty low, though these alumina particles are easily internalized by cells. The loss of cell viability and membrane integrity are dose-dependent and sample-dependent after alumina exposure. At concentrations lower than 100 microg/mL, no significant toxicity is observed for all alumina samples. When the concentration reaches 200 microg/mL, alumina treated cells begin to loss their activities. No culture period effect (up to 3 days) is observed. Very tiny soluble aluminum and the absorption of culture medium ingredients onto alumina particles do not affect the cell viability. Intracellular reactive oxygen species generation may contribute to the cytotoxicity of alumina particles at high concentration, but it does not induce the apoptosis of NSCs.

Aberrant neocortical DNA methylation has been suggested to be a pathophysiological contributor to psychotic disorders. Recently, a growth arrest and DNA-damage-inducible, beta (GADD45b) protein-coordinated DNA demethylation pathway, utilizing cytidine deaminases and thymidine glycosylases, has been identified in the brain. We measured expression of several members of this pathway in parietal cortical samples from the Stanley Foundation Neuropathology Consortium (SFNC) cohort. We find an increase in GADD45b mRNA and protein in patients with psychosis. In immunohistochemistry experiments using samples from the Harvard Brain Tissue Resource Center, we report an increased number of GADD45b-stained cells in prefrontal cortical layers II, III, and V in psychotic patients. Brain-derived neurotrophic factor IX (BDNF IXabcd) was selected as a readout gene to determine the effects of GADD45b expression and promoter binding. We find that there is less GADD45b binding to the BDNF IXabcd promoter in psychotic subjects. Further, there is reduced BDNF IXabcd mRNA expression, and an increase in 5-methylcytosine and 5-hydroxymethylcytosine at its promoter. On the basis of these results, we conclude that GADD45b may be increased in psychosis compensatory to its inability to access gene promoter regions.

Anopheles gambiae mosquitoes that transmit Plasmodium falciparum malaria use a series of olfactory cues present in human sweat to locate their hosts for a blood meal. Recognition of these odor cues occurs through the interplay of odorant receptors and odorant-binding proteins (OBPs) that bind to odorant molecules and transport and present them to the receptors. Recent studies have implicated potential heterodimeric interactions between two OBPs, OBP1 and OBP4, as important for perception of indole by the mosquito (Biessmann, H., Andronopoulou, E., Biessmann, M. R., Douris, V., Dimitratos, S. D., Eliopoulos, E., Guerin, P. M., Iatrou, K., Justice, R. W., Kröber, T., Marinotti, O., Tsitoura, P., Woods, D. F., and Walter, M. F.(2010) PLoS ONE 5, e9471; Qiao, H., He, X., Schymura, D., Ban, L., Field, L., Dani, F. R., Michelucci, E., Caputo, B., della Torre, A., Iatrou, K., Zhou, J. J., Krieger, J., and Pelosi, P.(2011) Cell. Mol. Life Sci. 68, 1799-1813). Here we present the 2.0 Å crystal structure of the OBP4-indole complex, which adopts a classical odorant-binding protein fold, with indole bound at one end of a central hydrophobic cavity. Solution-based NMR studies reveal that OBP4 exists in a molten globule state and binding of indole induces a dramatic conformational shift to a well ordered structure, and this leads to the formation of the binding site for OBP1. Analysis of the OBP4-OBP1 interaction reveals a network of contacts between residues in the OBP1 binding site and the core of the protein and suggests how the interaction of the two proteins can alter the binding affinity for ligands. These studies provide evidence that conformational ordering plays a key role in regulating heteromeric interactions between OBPs.

Activation of group II metabotropic glutamate receptors (mGlu2 and -3 receptors) has shown a potential antipsychotic activity, yet the underlying mechanism is only partially known. Altered epigenetic mechanisms contribute to the pathogenesis of schizophrenia and currently used medications exert chromatin remodeling effects. Here, we show that systemic injection of the brain-permeant mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268; 0.3-1 mg/kg i.p.) increased the mRNA and protein levels of growth arrest and DNA damage 45-β (Gadd45-β), a molecular player of DNA demethylation, in the mouse frontal cortex and hippocampus. Induction of Gadd45-β by LY379268 was abrogated by the mGlu2/3 receptor antagonist (2S)-2-amino-2-[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid (LY341495; 1 mg/kg i.p.). Treatment with LY379268 also increased the amount of Gadd45-β bound to specific promoter regions of reelin, brain-derived neurotrophic factor (BDNF), and glutamate decarboxylase-67 (GAD67). We directly assessed gene promoter methylation in control mice and in mice pretreated for 7 days with the methylating agent methionine (750 mg/kg i.p.). Both single and repeated injections with LY379268 reduce cytosine methylation in the promoters of the three genes, although the effect on the GAD67 was significant only in response to repeated injections. Single and repeated treatment with LY379268 could also reverse the defect in social interaction seen in mice pretreated with methionine. The action of LY379268 on Gadd45-β was mimicked by valproate and clozapine but not haloperidol. These findings show that pharmacological activation of mGlu2/3 receptors has a strong impact on the epigenetic regulation of genes that have been linked to the pathophysiology of schizophrenia.

Nicotine improves cognitive performance and attention in both experimental animals and in human subjects, including patients affected by neuropsychiatric disorders. However, the specific molecular mechanisms underlying nicotine-induced behavioral changes remain unclear. We have recently shown in mice that repeated injections of nicotine, which achieve plasma concentrations comparable to those reported in high cigarette smokers, result in an epigenetically induced increase of glutamic acid decarboxylase 67 (GAD(67)) expression. Here we explored the impact of synthetic α(4)β(2) and α(7) nAChR agonists on GABAergic epigenetic parameters. Varenicline (VAR), a high-affinity partial agonist at α(4)β(2) and a lower affinity full agonist at α(7) neuronal nAChR, injected in doses of 1-5 mg/kg/s.c. twice daily for 5 days, elicited a 30-40% decrease of cortical DNA methyltransferase (DNMT)1 mRNA and an increased expression of GAD(67) mRNA and protein. This upregulation of GAD(67) was abolished by the nAChR antagonist mecamylamine. Furthermore, the level of MeCP(2) binding to GAD(67) promoters was significantly reduced following VAR administration. This effect was abolished when VAR was administered with mecamylamine. Similar effects on cortical DNMT1 and GAD(67) expression were obtained after administration of A-85380, an agonist that binds to α(4)β(2) but has negligible affinity for α(3)β(4) or α(7) subtypes containing nAChR. In contrast, PNU-282987, an agonist of the homomeric α(7) nAChR, failed to decrease cortical DNMT1 mRNA or to induce GAD(67) expression. The present study suggests that the α(4)β(2) nAChR agonists may be better suited to control the epigenetic alterations of GABAergic neurons in schizophrenia than the α(7) nAChR agonists.

Continuing improvements in analytical technology along with an increased interest in performing comprehensive, quantitative metabolic profiling, is leading to increased interest pressures within the metabolomics community to develop centralized metabolite reference resources for certain clinically important biofluids, such as cerebrospinal fluid, urine and blood. As part of an ongoing effort to systematically characterize the human metabolome through the Human Metabolome Project, we have undertaken the task of characterizing the human serum metabolome. In doing so, we have combined targeted and non-targeted NMR, GC-MS and LC-MS methods with computer-aided literature mining to identify and quantify a comprehensive, if not absolutely complete, set of metabolites commonly detected and quantified (with today's technology) in the human serum metabolome. Our use of multiple metabolomics platforms and technologies allowed us to substantially enhance the level of metabolome coverage while critically assessing the relative strengths and weaknesses of these platforms or technologies. Tables containing the complete set of 4229 confirmed and highly probable human serum compounds, their concentrations, related literature references and links to their known disease associations are freely available at http://www.serummetabolome.ca.

The methylation and demethylation of CpG dinucleotides that are embedded in promoters play an important role in controlling gene transcription. In the mammalian brain, CpG promoter methylation is a postreplicative process mediated by a group of DNA methyltransferases (DNMT), such as DNMT1 and DNMT3a, DNMT3b. Several studies demonstrate that in addition to DNMTs, promoter methylation in the brain can be regulated by a putative DNA demethylation process that specifically removes the methyl group from the carbon-5 of cytosines. To test the existence of a possible active DNA demethylation activity in postmitotic neuronal or glial cells, we incubated an SssI methylated mouse reelin (Reln) promoter fragment (-720 to +140) with nuclear extracts from the mouse frontal cortex (FC). We observed the presence of DNA demethylation activity, which was increased in FC nuclear extracts from mice treated with valproate (VPA, 2.2 mmol/kg, twice a day for 3 days). VPA not only reduces anxiety, and cognitive deficits, and other symptoms in bipolar disorder (BP) disorder and schizophrenia (SZ) patients but also upregulates Reln and glutamic acid decarboxylase 67 (Gad67) mRNA/protein expression by reducing the methylation of their promoters. We believe that the identification of an enzyme in brain that facilitates DNA-demethylation and an understanding of how drugs induce DNA demethylation are crucial to progress in a new line of pharmacological interventions to treat neurodevelopment, neuropsychiatric, and neurodegenerative diseases.