Lipidomics Reveals Early Metabolic Changes in Subjects with Schizophrenia: Effects of Atypical Antipsychotics

There is a critical need for mapping early metabolic changes in schizophrenia to capture failures in regulation of biochemical pathways and networks. This information could provide valuable insights about disease mechanisms, trajectory of disease progression, and diagnostic biomarkers. We used a lipidomics platform to measure individual lipid species in 20 drug-naïve patients with a first episode of schizophrenia (FE group), 20 patients with chronic schizophrenia that had not adhered to prescribed medications (RE group), and 29 race-matched control subjects without schizophrenia. Lipid metabolic profiles were evaluated and compared between study groups and within groups before and after treatment with atypical antipsychotics, risperidone and aripiprazole. Finally, we mapped lipid profiles to n3 and n6 fatty acid synthesis pathways to elucidate which enzymes might be affected by disease and treatment. Compared to controls, the FE group showed significant down-regulation of several n3 polyunsaturated fatty acids (PUFAs), including 20:5n3, 22:5n3, and 22:6n3 within the phosphatidylcholine and phosphatidylethanolamine lipid classes. Differences between FE and controls were only observed in the n3 class PUFAs; no differences where noted in n6 class PUFAs. The RE group was not significantly different from controls, although some compositional differences within PUFAs were noted. Drug treatment was able to correct the aberrant PUFA levels noted in FE patients, but changes in re patients were not corrective. Treatment caused increases in both n3 and n6 class lipids. These results supported the hypothesis that phospholipid n3 fatty acid deficits are present early in the course of schizophrenia and tend not to persist throughout its course. These changes in lipid metabolism could indicate a metabolic vulnerability in patients with schizophrenia that occurs early in development of the disease.     

Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background

The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.

Results

We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/C^CDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.

Conclusion

Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.     

Protein synthesis in adult skeletal muscle after tenotomy: responses to fasting and insulin infusion.

Muscle growth was established in specific muscles in the hindlimb of adult female rats by tenotomy of the gastrocnemius muscle. Seven days after surgery there was an increase in the wet weight of the soleus (Sol) and plantaris (P) muscles and a decrease in that of the gastrocnemius (G) muscle from the tenotomized limb compared with the respective control muscles from the contralateral limb from the same animal. In all three muscles there was a significant increase in the fractional rate of protein synthesis (ks) in the muscles from the tenotomized limb above the rate of the respective control muscles. In contrast, the extensor digitorum longus (EDL) muscle showed no change in wet weight or ks 7 days after tenotomy of G. Fasting for 12 or 36 h had no significant effect on ks in G, P, or Sol muscles from either the control or tenotomized limbs. In EDL from the control limb, both fasting periods resulted in a significant decrease in ks, although this effect was not seen in the EDL from the tenotomized limbs of the same animals. A subsequent 30-min insulin infusion was similarly ineffectual in G, P, and Sol, with its only effect evident in the EDL from the control limb, where it was sufficient to reverse the decreased ks resulting from the fasting, even though after 36 h fasting the reversal was only partial.     

Cytochrome P450 3A4-mediated oxidative conversion of a cyano to an amide group in the metabolism of pinacidil

The conversion of nitriles to amides is generally considered to be a hydrolytic process that does not involve redox chemistry. We demonstrate here that cytochrome P450 (CYP) is responsible for the conversion of the cyano group of pinacidil to the corresponding amide. The reaction in human liver microsomes was NADPH-dependent and was nearly completely inhibited by an anti-CYP3A4 antibody. Incubations of pinacidil with recombinant CYP enzymes confirm that CYP3A4 is the principal catalyst of this reaction. The kinetics of pinacidil amide formation by CYP3A4 yielded an apparent K(m) of 452 +/- 33 microM and k(cat) of 0.108 min(-1) (k(cat)/K(m) = 0.238 mM(-1).min(-1)). Incubation of pinacidil with CYP3A4 in the presence of (18)O(2) or H(2)(18)O showed that the amide carbonyl oxygen derived exclusively from molecular oxygen. The CYP3A4-mediated reaction also was supported by hydrogen peroxide when incubations were carried out in the absence of cytochrome P450 reductase and NADPH. The reaction can be explained by a nucleophilic attack of a deprotonated ferric peroxide intermediate (Fe(3+)-O-O(-)) on the carbon atom of the -C triple bond N triple bond to form an Enz-Fe(III)-O-O-C(=NH)R intermediate, followed by cleavage of the O-O bond to give pinacidil amide. This nucleophilic addition of an Fe(3+)-O-O(-) intermediate to a -C=N pi-bond in a P450 system resembles the analogous reaction catalyzed by the nitric oxide synthases.     

ERK2 mitogen-activated protein kinase binding, phosphorylation, and regulation of the PDE4D cAMP-specific phosphodiesterases. The involvement of COOH-terminal docking sites and NH2-terminal UCR regions

The cAMP-specific phosphodiesterase family 4, subfamily D, isoform 3 (PDE4D3) is shown to have FQF and KIM docking sites for extracellular signal-regulated kinase 2 (ERK2) (p42(MAPK)). These straddle the target residue, Ser(579), for ERK2 phosphorylation of PDE4D3. Mutation of either or both of these docking sites prevented ERK2 from being co-immunoprecipitated with PDE4D3, ablated the ability of epidermal growth factor to inhibit PDE4D3 through ERK2 action in transfected COS cells, and attenuated the ability of ERK2 to phosphorylate PDE4D3 in vitro. The two conserved NH(2)-terminal blocks of sequence, called upstream conserved regions 1 and 2 (UCR1 and UCR2), that characterize PDE4 long isoforms, are proposed to amplify the small, inherent inhibitory effect that ERK2 phosphorylation exerts on the PDE4D catalytic unit. In contrast to this, the lone intact UCR2 region found in PDE4D1 directs COOH-terminal ERK2 phosphorylation to cause the activation of this short isoform. From the analysis of PDE4D3 truncates, it is suggested that UCR1 and UCR2 provide a regulatory signal integration module that serves to orchestrate the functional consequences of ERK2 phosphorylation. The PDE4D gene thus encodes a series of isoenzymes that are either inhibited or activated by ERK2 phosphorylation and thereby offers the potential for ERK2 activation either to increase or decrease cAMP levels in cellular compartments.     

The structure of the human RNase H2 complex defines key interaction interfaces relevant to enzyme function and human disease

Ribonuclease H2 (RNase H2) is the major nuclear enzyme involved in the degradation of RNA/DNA hybrids and removal of ribonucleotides misincorporated in genomic DNA. Mutations in each of the three RNase H2 subunits have been implicated in a human auto-inflammatory disorder, Aicardi-Goutières Syndrome (AGS). To understand how mutations impact on RNase H2 function we determined the crystal structure of the human heterotrimer. In doing so, we correct several key regions of the previously reported murine RNase H2 atomic model and provide biochemical validation for our structural model. Our results provide new insights into how the subunits are arranged to form an enzymatically active complex. In particular, we establish that the RNASEH2A C terminus is a eukaryotic adaptation for binding the two accessory subunits, with residues within it required for enzymatic activity. This C-terminal extension interacts with the RNASEH2C C terminus and both are necessary to form a stable, enzymatically active heterotrimer. Disease mutations cluster at this interface between all three subunits, destabilizing the complex and/or impairing enzyme activity. Altogether, we locate 25 out of 29 residues mutated in AGS patients, establishing a firm basis for future investigations into disease pathogenesis and function of the RNase H2 enzyme.     

Most ultraviolet irradiation induced mutations in the nematode Caenorhabditis elegans are chromosomal rearrangements

In this study we have determined the utility of 254-nm ultraviolet light (UV) as a mutagenic tool in C. elegans. We have demonstrated that irradiation of adult hermaphrodites provides a simple method for the induction of heritable chromosomal rearrangements. A screening protocol was employed that identifies either recessive lethal mutations in the 40 map unit region balanced by the translocation eT1(III;V), or unc-36(III) duplications. Mutations were recovered in 3% of the chromosomes screened after a dose of 120 J/m2. This rate resembles that for 1500 R gamma-ray-induced mutations selected in a similar manner. The mutations were classified either as lethals [mapping to Linkage Group (LG)III or LGV] or as putative unc-36 duplications. In contrast to the majority of UV-induced mutations analysed in microorganisms, we found that a large fraction of the C. elegans UV-induced mutations are not simple intragenic lesions, but are deficiencies for more than one adjacent gene or more complex events. Preliminary evidence for this conclusion came from the high frequency of mutations that had a dominant effect causing reduced numbers of adult progeny. Subsequently 6 out of 9 analysed LGV mutations were found to be deficiencies. Other specific rearrangements also identified were: one translocation, sT5(II;III), and two unc-36 duplications, sDp8 and sDp9. It was concluded that UV irradiation can easily be used as an additional tool for the analysis of C. elegans chromosomes, and that C. elegans should prove to be a useful organism in which to study the mechanisms whereby UV acts as a mutagen in cells of complex eukaryotes.