IMPA1 is essential for embryonic development and lithium-like pilocarpine sensitivity.

Lithium has been the standard pharmacological treatment for bipolar disorder over the last 50 years; however, the molecular targets through which lithium exerts its therapeutic effects are still not defined. We characterized the phenotype of mice with a dysfunctional IMPA1 gene (IMPA1-/-) to study the in vivo physiological functions of IMPA1, in general, and more specifically its potential role as a molecular target in mediating lithium-dependent physiological effects. Homozygote IMPA1-/- mice died in utero between days 9.5 and 10.5 post coitum (p.c.) demonstrating the importance of IMPA1 in early embryonic development. Intriguingly, the embryonic lethality could be reversed by myo-inositol supplementation via the pregnant mothers. In brains of adult IMPA1-/- mice, IMPase activity levels were found to be reduced (up to 65% in hippocampus); however, inositol levels were not found to be altered. Behavioral analysis of the IMPA1-/- mice indicated an increased motor activity in both the open-field test and the forced-swim test as well as a strongly increased sensitivity to pilocarpine-induced seizures, the latter supporting the idea that IMPA1 represents a physiologically relevant target for lithium. In conclusion the IMPA1-/- mouse represents a novel model to study inositol homeostasis, and indicates that genetic inactivation of IMPA1 can mimic some actions of lithium.

I/NI-calls for the exclusion of non-informative genes: a highly effective filtering tool for microarray data.

MOTIVATION: DNA microarray technology typically generates many measurements of which only a relatively small subset is informative for the interpretation of the experiment. To avoid false positive results, it is therefore critical to select the informative genes from the large noisy data before the actual analysis. Most currently available filtering techniques are supervised and therefore suffer from a potential risk of overfitting. The unsupervised filtering techniques, on the other hand, are either not very efficient or too stringent as they may mix up signal with noise. We propose to use the multiple probes measuring the same target mRNA as repeated measures to quantify the signal-to-noise ratio of that specific probe set. A Bayesian factor analysis with specifically chosen prior settings, which models this probe level information, is providing an objective feature filtering technique, named informative/non-informative calls (I/NI calls). RESULTS: Based on 30 real-life data sets (including various human, rat, mice and Arabidopsis studies) and a spiked-in data set, it is shown that I/NI calls is highly effective, with exclusion rates ranging from 70% to 99%. Consequently, it offers a critical solution to the curse of high-dimensionality in the analysis of microarray data. AVAILABILITY: This filtering approach is publicly available as a function implemented in the R package FARMS (www.bioinf.jku.at/software/farms/farms.html).

Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain.

Uptake of L-glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (VGLUTs). Three transporters (VGLUT1-VGLUT3) are expressed in the mammalian CNS, with partial overlapping expression patterns, and VGLUT2 is the most abundantly expressed paralog in the thalamus, midbrain, and brainstem. Previous studies have shown that VGLUT1 is necessary for glutamatergic transmission in the hippocampus, but the role of VGLUT2 in excitatory transmission is unexplored in glutamatergic neurons and in vivo. We examined the electrophysiological and behavioral consequences of loss of either one or both alleles of VGLUT2. We show that targeted deletion of VGLUT2 in mice causes perinatal lethality and a 95% reduction in evoked glutamatergic responses in thalamic neurons, although hippocampal synapses function normally. Behavioral analysis of heterozygous VGLUT2 mice showed unchanged motor function, learning and memory, acute nociception, and inflammatory pain, but acquisition of neuropathic pain, maintenance of conditioned taste aversion, and defensive marble burying were all impaired. Reduction or loss of VGLUT2 in heterozygous and homozygous VGLUT2 knock-outs led to a graded reduction in the amplitude of the postsynaptic response to single-vesicle fusion in thalamic neurons, indicating that the vesicular VGLUT content is critically important for quantal size and demonstrating that VGLUT2-mediated reduction of excitatory drive affects specific forms of sensory processing.

p53-independent regulation of p21Waf1/Cip1 expression and senescence by Chk2.

The Chk2 kinase is a tumor suppressor and key component of the DNA damage checkpoint response that encompasses cell cycle arrest, apoptosis, and DNA repair. It has also been shown to have a role in replicative senescence resulting from dysfunctional telomeres. Some of these functions are at least partially exerted through activation of the p53 transcription factor. High-level expression of virally transduced Chk2 in A549 human lung carcinoma cells led to arrested proliferation, apoptosis, and senescence. These were accompanied by various molecular events, including p21(Waf1/Cip1) (p21) transcriptional induction, consistent with p53 activation. However, Chk2-dependent senescence and p21 transcriptional induction also occurred in p53-defective SK-BR-3 (breast carcinoma) and HaCaT (immortalized keratinocyte) cells. Small interfering RNA-mediated knockdown of p21 in p53-defective cells expressing Chk2 resulted in a decrease in senescent cells. These results revealed a p53-independent role for Chk2 in p21 induction and senescence that may contribute to tumor suppression and genotoxic treatment outcome.

Gene expression profiles highlight adaptive brain mechanisms in corticotropin releasing factor overexpressing mice.

Corticotropin-releasing factor (CRF) plays an important role in mediating central and peripheral responses to stress. Alterations in CRF system activity have been linked to a number of psychiatric disorders, including anxiety and depression. Aim of this study was to elucidate homeostatic mechanisms induced by lifelong elevated CRF levels in the brain. We therefore profiled gene expression in several brain areas of transgenic mice overexpressing CRF (CRF-OE), a model for chronic stress. Several genes showed altered expression levels in CRF-OE mice when compared to their wild type littermates and were confirmed by quantitative PCR. Differences in gene expression profiles revealed the presence of previously unrecognized homeostatic mechanisms in CRF-OE animals. These included changes in glucocorticoid signaling, as exemplified by changes in 11beta-hydroxysteroid dehydrogenase type 1, FK506 binding protein 5 and serum/glucocorticoid kinase. Alterations in expression of genes involved in myelination (myelin, myelin-associated glycoprotein), cell proliferation and extracellular matrix formation (Edg2, Fgfr2, decorin, brevican) suggest changes in the dynamics of neurogenesis in CRF-OE. Pronounced changes in neurotensin (NT) receptors 1 and 2 mRNA were identified. Overall downregulation of NT receptors in CRF-OE animal was substantiated by receptor binding studies. Pronounced neurotensin receptor downregulation was observed for NT type 1 receptors in limbic brain areas, suggesting that NT could be implicated in some of the effects attributed to CRF overexpression. These data show that lifelong exposure to excessive CRF leads to adaptive changes in the brain which could play a role in some of the behavioral and physiological alterations seen in these animals.

Sensory deficits in mice hypomorphic for a mammalian homologue of unc-53.

The migration of cells and the extension of cellular processes along pathways to their defined destinations are crucial in the development of higher organisms. Caenorhabditis elegans unc-53 plays an important role in cell migration and the outgrowth of cellular processes such as axons. To gain further insight into the biological function of unc53H2, a recently identified mammalian homologue of unc-53, we have generated mice carrying a mutation of unc53H2 and provide evidence that unc53H2 is involved in neuronal development and, more specifically, the development of different sensory systems. The unc53H2 hypomorphic mouse showed a general impaired acuity of several sensory systems (olfactory, auditory, visual and pain sensation) which in case of the visual system was corroborated by the morphological observation of hypoplasia of the optic nerve. We hypothesize that in analogy with its C. elegans homologue, unc53H2 may play a role in the processes of cellular outgrowth and migration.

Altered gastrointestinal and metabolic function in the GPR39-obestatin receptor-knockout mouse.

BACKGROUND & AIMS: The G-protein-coupled receptor GPR39 is a member of a family that includes the receptors for ghrelin and motilin. Recently the peptide obestatin was identified as a natural ligand for GPR39. The objective of this study was to gain insight into the biological function of the GPR39 receptor. METHODS: GPR39(-/-) mice were generated and analyzed. RESULTS: Endogenous GPR39 expression was detected in the brain (septum-amygdala) and the gastrointestinal system (parietal cells, enterocytes, neurons, and pancreas). Gastric emptying of a solid meal (measured by the (14)C octanoic breath test) in GPR39(-/-) mice was accelerated significantly with a gastric half-emptying time of 49.5 +/- 2.2 minutes compared with 86.9 +/- 8.4 minutes in GPR39(+/+) mice. A more effective expulsion of distally located pellets (30%-75% of length) was observed in the colon of GPR39(-/-) mice. Four hours after pylorus ligation, the volume of gastric secretion was increased significantly (GPR39(-/-): 638 +/- 336 microL; GPR39(+/+): 225 +/- 170 microL), but gastric acid secretion was unchanged. The mature body weight and body fat composition of GPR39(-/-) mice was significantly higher compared with GPR39(+/+) mice, but this was not related to hyperphagia because 24-hour food intake did not differ between both genotypes. In contrast, deficiency of the GPR39 receptor led to reduced hyperphagia after fasting. The cholesterol levels were increased significantly in the GPR39(-/-) mice. CONCLUSIONS: Our data partially confirm and extend the described in vivo effects of obestatin and suggest that this peptide plays a functional role in the regulation of gastrointestinal and metabolic function through interaction with the GPR39 receptor.

Graphical exploration of gene expression data: a comparative study of three multivariate methods.

This article describes three multivariate projection methods and compares them for their ability to identify clusters of biological samples and genes using real-life data on gene expression levels of leukemia patients. It is shown that principal component analysis (PCA) has the disadvantage that the resulting principal factors are not very informative, while correspondence factor analysis (CFA) has difficulties interpreting distances between objects. Spectral map analysis (SMA) is introduced as an alternative approach to the analysis of microarray data. Weighted SMA outperforms PCA, and is at least as powerful as CFA, in finding clusters in the samples, as well as identifying genes related to these clusters. SMA addresses the problem of data analysis in microarray experiments in a more appropriate manner than CFA, and allows more flexible weighting to the genes and samples. Proper weighting is important, since it enables less reliable data to be down-weighted and more reliable information to be emphasized.

Transcriptional response to corticotropin-releasing factor in AtT-20 cells.

Corticotropin-releasing factor (CRF) plays a central role in the regulation of the hypothalamic-pituitary-adrenal axis, mediating endocrine and behavioral responses to various stressors. Two high-affinity receptors for CRF have been described. Although many of the intracellular signaling pathways activated by CRF have been studied extensively, our knowledge of transcriptional responses downstream of the CRF receptor 1 (CRFR1) is still limited. To elucidate gene networks regulated by CRF and CRFR1, we applied microarray technology to explore transcriptional response to CRF stimulation. Therefore, mouse pituitary-derived AtT-20 cells were exposed continuously to CRF either in the presence or absence of the specific CRFR1 antagonist R121919. Transcriptional responses to different treatments were studied in a time course ranging from 0.5 to 24 h. Microarray data were analyzed using classic microarray data analysis tools such as correspondence factor analysis, cluster analysis, and fold-change filtering. Furthermore, spectral map analysis was applied, a recently introduced unsupervised multivariate analysis method. A broad and transient transcriptional response to CRF was identified that could be blocked by the antagonist. This way, several known CRF-induced target genes and novel CRF responsive genes were identified. These include transcription factors such as cAMP-responsive element modulator (7x increased), secreted peptides such as cholecystokinin (1.5x), and proteins involved in modulating intracellular signaling, such as regulator of G-protein signaling 2 (11x). Up-regulation of many of these genes can be explained as negative feedback, attenuating CRF-activated pathways. In addition, spectral map analysis proved to be a promising new tool for microarray data analysis.