VHL-deficiency leads to reductive stress in renal cells.

Heritable renal cancer syndromes (RCS) are associated with numerous chromosomal alterations including inactivating mutations in von Hippel-Lindau (VHL) gene. Here we identify a novel aspect of the phenotype in VHL-deficient human renal cells. We call it reductive stress as it is characterised by increased NADH/NAD+ ratio that is associated with impaired cellular respiration, impaired CAC activity, upregulation of reductive carboxylation of glutamine and accumulation of lipid droplets in VHL-deficient cells. Reductive stress was mitigated by glucose depletion and supplementation with pyruvate or resazurin, a redox-reactive agent. This study demonstrates for the first time that reductive stress is a part of the phenotype associated with VHL-deficiency in renal cells and indicates that the reversal of reductive stress can augment respiratory activity and CAC activity, suggesting a strategy for altering the metabolic profile of VHL-deficient tumours.

Hypothermia Alleviates Reductive Stress, a Root Cause of Ischemia Reperfusion Injury.

Ischemia reperfusion injury is common in transplantation. Previous studies have shown that cooling can protect against hypoxic injury. To date, the protective effects of hypothermia have been largely associated with metabolic suppression. Since kidney transplantation is one of the most common organ transplant surgeries, we used human-derived renal proximal tubular cells (HKC8 cell line) as a model of normal renal cells. We performed a temperature titration curve from 37 °C to 22 °C and evaluated cellular respiration and molecular mechanisms that can counteract the build-up of reducing equivalents in hypoxic conditions. We show that the protective effects of hypothermia are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component), with the highest overlap between activating and suppressing mechanisms emerging in the window of mild hypothermia (32 °C). Hypothermia decreased hypoxia-induced rise in the extracellular lactate:pyruvate ratio, increased ATP/ADP ratio and mitochondrial content, normalized lipid content, and improved the recovery of respiration after anoxia. Importantly, it was observed that in contrast to mild hypothermia, moderate and deep hypothermia interfere with HIF1 (hypoxia inducible factor 1)-dependent HRE (hypoxia response element) induction in hypoxia. This work also demonstrates that hypothermia alleviates reductive stress, a conceptually novel and largely overlooked phenomenon at the root of ischemia reperfusion injury.

Proline synthesis through PYCR1 is required to support cancer cell proliferation and survival in oxygen-limiting conditions.

The demands of cancer cell proliferation alongside an inadequate angiogenic response lead to insufficient oxygen availability in the tumor microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing potential produced through tricarboxylic acid (TCA) cycle activity and mitochondrial respiration are functionally linked. As the oxidizing activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidizing NADH with the synthesis of proline as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting continued TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

Hypothermia augments stress response in mammalian cells.

Mild hypothermia (32 °C) is routinely used in medical practice to alleviate hypoxic ischemic damage, however, the mechanisms that underlie its protective effects remain uncertain. Using a systems approach based on genome-wide expression screens, reporter assays and biochemical studies, we find that cellular hypothermia response is associated with the augmentation of major stress-inducible transcription factors Nrf2 and HIF1Α affecting the antioxidant system and hypoxia response pathways, respectively. At the same time, NF-κB, a transcription factor involved in the control of immune and inflammatory responses, was not induced by hypothermia. Furthermore, mild hypothermia did not trigger unfolded protein response. Lower temperatures (27 °C and 22 °C) did not activate Nrf2 and HIF1A pathways as efficiently as mild hypothermia. Current findings are discussed in the context of the thermodynamic hypothesis of therapeutic hypothermia. We argue that the therapeutic effects are likely to stem both from metabolic suppression (inhibitory component) and augmentation of stress tolerance (activating component). We argue that systems coping with cellular stressors are plausible targets of therapeutic hypothermia and deserve more attention in clinical hypothermia research.

Exenatide Is an Effective Antihyperglycaemic Agent in a Mouse Model of Wolfram Syndrome 1.

Wolfram syndrome 1 is a very rare monogenic disease resulting in a complex of disorders including diabetes mellitus. Up to now, insulin has been used to treat these patients. Some of the monogenic forms of diabetes respond preferentially to sulphonylurea preparations. The aim of the current study was to elucidate whether exenatide, a GLP-1 receptor agonist, and glipizide, a sulphonylurea, are effective in a mouse model of Wolfram syndrome 1. Wolframin-deficient mice were used to test the effect of insulin secretagogues. Wolframin-deficient mice had nearly normal fasting glucose levels but developed hyperglycaemia after glucose challenge. Exenatide in a dose of 10 µg/kg lowered the blood glucose level in both wild-type and wolframin-deficient mice when administered during a nonfasted state and during the intraperitoneal glucose tolerance test. Glipizide (0.6 or 2 mg/kg) was not able to reduce the glucose level in wolframin-deficient animals. In contrast to other groups, wolframin-deficient mice had a lower insulin-to-glucose ratio during the intraperitoneal glucose tolerance test, indicating impaired insulin secretion. Exenatide increased the insulin-to-glucose ratio irrespective of genotype, demonstrating the ability to correct the impaired insulin secretion caused by wolframin deficiency. We conclude that GLP-1 agonists may have potential in the treatment of Wolfram syndrome-related diabetes.

Deletion of the Wolfram syndrome-related gene Wfs1 results in increased sensitivity to ethanol in female mice.

Wolfram syndrome, induced by mutation in WFS1 gene, increases risk of developing mood disorders in humans. In mice, Wfs1 deficiency cause higher anxiety-like behaviour and increased response to anxiolytic-like effect of diazepam, a GABAA receptor agonist. As GABAergic system is also target for ethanol, we analysed its anxiolytic-like and sedative properties in Wfs1-deficient mice using elevated plus-maze test and tests measuring locomotor activity and coordination, respectively. Additionally loss of righting reflex test was conducted to study sedative/hypnotic properties of ethanol, ketamine and pentobarbital. To evaluate pharmacokinetics of ethanol in mice enzymatic colour test was used. Finally, gene expression of alpha subunits of GABAA receptors following ethanol treatment was studied by real-time-PCR. Compared to wild-types, Wfs1-deficient mice were more sensitive to ethanol-induced anxiolytic-like effect, but less responsive to impairment of motor coordination. Ethanol and pentobarbital, but not ketamine, caused longer duration of hypnosis in Wfs1-deficient mice. The expression of Gabra2 subunit at 30 minutes after ethanol injection was significantly increased in the frontal cortex of Wfs1-deficient mice as compared to respective vehicle-treated mice. For the temporal lobe, similar change in Gabra2 mRNA occurred at 60 minutes after ethanol treatment in Wfs1-deficient mice. No changes were detected in Gabra1 and Gabra3 mRNA following ethanol treatment. Taken together, increased anxiolytic-like effect of ethanol in Wfs1-deficient mice is probably related to altered Gabra2 gene expression. Increased anti-anxiety effect of GABAA receptor agonists in the present work and earlier studies (Luuk et al., 2009) further suggests importance of Wfs1 gene in the regulation of emotional behaviour.

Lsamp is implicated in the regulation of emotional and social behavior by use of alternative promoters in the brain.

Limbic system-associated membrane protein (LSAMP) is a neural cell adhesion molecule involved in neurite formation and outgrowth. The purpose of the present study was to characterize the distribution of alternatively transcribed Lsamp isoforms in the mouse brain and its implications on the regulation of behavior. Limbic system-associated membrane protein 1b transcript was visualized by using a mouse strain expressing beta-galactosidase under the control of Lsamp 1b promoter. The distribution of Lsamp 1a transcript and summarized expression of the Lsamp transcripts was investigated by non-radioactive in situ RNA hybridization analysis. Cross-validation was performed by using radioactive in situ hybridization with oligonucleotide probes. Quantitative RT-PCR was used to study correlations between the expression of Lsamp isoforms and behavioral parameters. The expression pattern of two promoters differs remarkably from the developmental initiation at embryonic day 12.5. Limbic system-associated membrane protein 1a promoter is active in "classic" limbic structures where the hippocampus and amygdaloid area display the highest expression. Promoter 1b is mostly active in the thalamic sensory nuclei and cortical sensory areas, but also in areas that regulate stress and arousal. Higher levels of Lsamp 1a transcript had significant correlations with all of the measures indicating higher trait anxiety in the elevated plus-maze test. Limbic system-associated membrane protein transcript levels in the hippocampus and ventral striatum correlated with behavioral parameters in the social interaction test. The data are in line with decreased anxiety and alterations in social behavior in Lsamp-deficient mice. We propose that Lsamp is involved in emotional and social operating systems by complex regulation of two alternative promoters.

Effect of light on global gene expression in the neuroglobin-deficient mouse retina.

Several previous studies have raised controversy over the functional role of neuroglobin (Ngb) in the retina. Certain studies indicate a significant impact of Ngb on retinal physiology, whereas others are conflicting. The present is an observational study that tested the effect of Ngb deficiency on gene expression in dark- and light-adapted mouse retinas. Large-scale gene expression profiling was performed using GeneChip® Mouse Exon 1.0 ST arrays and the results were compared to publicly available data sets. The lack of Ngb was found to have a minor effect on the light-induced retinal gene expression response. In addition, there was no increase in the expression of marker genes associated with hypoxia, endoplasmic reticulum-stress and oxidative stress in the Ngb-deficient retina. By contrast, several genes were identified that appeared to be differentially expressed between the genotypes when the effect of light was ignored. The present study indicates that Ngb deficiency does not lead to major alternations in light-dependent gene expression response, but leads to subtle systemic differences of a currently unknown functional significance.

Initiation and developmental dynamics of Wfs1 expression in the context of neural differentiation and ER stress in mouse forebrain.

Wolframin (Wfs1) is a membrane glycoprotein that resides in the endoplasmic reticulum (ER) and regulates cellular Ca(2+) homeostasis. In pancreas Wfs1 attenuates unfolded protein response (UPR) and protects cells from apoptosis. Loss of Wfs1 function results in Wolfram syndrome (OMIM 222300) characterized by early-onset diabetes mellitus, progressive optic atrophy, diabetes insipidus, deafness, and psychiatric disorders. Similarly, Wfs1-/- mice exhibit diabetes and increased basal anxiety. In the adult central nervous system Wfs1 is prominent in central extended amygdala, striatum and hippocampus, brain structures largely involved in behavioral adaptation of the organism. Here, we describe the initiation pattern of Wfs1 expression in mouse forebrain using mRNA in situ hybridization and compare it with Synaptophysin (Syp1), a gene encoding synaptic vesicle protein widely used as neuronal differentiation marker. We show that the expression of Wfs1 starts during late embryonic development in the dorsal striatum and amygdala, then expands broadly at birth, possessing several transitory regions during maturation. Syp1 expression precedes Wfs1 and it is remarkably upregulated during the period of Wfs1 expression initiation and maturation, suggesting relationship between neural activation and Wfs1 expression. Using in situ hybridization and quantitative real-time PCR we show that UPR-related genes (Grp78, Grp94, and Chop) display dynamic expression in the perinatal brain when Wfs1 is initiated and their expression pattern is not altered in the brain lacking functional Wfs1.

Estimating differential expression from multiple indicators.

Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR.

Wfs1-deficient mice display altered function of serotonergic system and increased behavioral response to antidepressants.

It has been shown that mutations in the WFS1 gene make humans more susceptible to mood disorders. Besides that, mood disorders are associated with alterations in the activity of serotonergic and noradrenergic systems. Therefore, in this study, the effects of imipramine, an inhibitor of serotonin (5-HT) and noradrenaline (NA) reuptake, and paroxetine, a selective inhibitor of 5-HT reuptake, were studied in tests of behavioral despair. The tail suspension test (TST) and forced swimming test (FST) were performed in Wfs1-deficient mice. Simultaneously, gene expression and monoamine metabolism studies were conducted to evaluate changes in 5-HT- and NA-ergic systems of Wfs1-deficient mice. The basal immobility time of Wfs1-deficient mice in TST and FST did not differ from that of their wild-type littermates. However, a significant reduction of immobility time in response to lower doses of imipramine and paroxetine was observed in homozygous Wfs1-deficient mice, but not in their wild-type littermates. In gene expression studies, the levels of 5-HT transporter (SERT) were significantly reduced in the pons of homozygous animals. Monoamine metabolism was assayed separately in the dorsal and ventral striatum of naive mice and mice exposed for 30 min to brightly lit motility boxes. We found that this aversive challenge caused a significant increase in the levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-HT, in the ventral and dorsal striatum of wild-type mice, but not in their homozygous littermates. Taken together, the blunted 5-HT metabolism and reduced levels of SERT are a likely reason for the elevated sensitivity of these mice to the action of imipramine and paroxetine. These changes in the pharmacological and neurochemical phenotype of Wfs1-deficient mice may help to explain the increased susceptibility of Wolfram syndrome patients to depressive states.

Evidence for impaired function of dopaminergic system in Wfs1-deficient mice.

Immunohistological studies suggest abundant expression of Wfs1 protein in neurons and nerve fibers that lie in the vicinity of dopaminergic (DA-ergic) fibers and neurons. Therefore, we sought to characterize the function of DA-ergic system in Wfs1-deficient mice. In wild-type mice, amphetamine, an indirect agonist of DA, caused significant hyperlocomotion and increase in tissue DA levels in the dorsal and ventral striatum. Both effects of amphetamine were significantly blunted in homozygous Wfs1-deficient mice. Motor stimulation caused by apomorphine, a direct DA receptor agonist, was somewhat stronger in Wfs1-deficient mice compared to their wild-type littermates. However, apomorphine caused a similar reduction in levels of DA metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) in the dorsal and ventral striatum in all genotypes. Behavioral sensitization to repeated treatment with amphetamine (2.5 mg/kg) was observed in wild-type, but not in Wfs1-deficient mice. The expression of DA transporter gene (Dat) mRNA was significantly lower in the midbrain of male and female homozygous mice compared to wild-type littermates. Altogether, the blunted effects of amphetamine and the reduced gene expression of DA transporter are probably indicative of an impaired functioning of the DA-ergic system in Wfs1-deficient mice.

Circadian rhythms and food anticipatory behavior in Wfs1-deficient mice.

The dorsomedial hypothalamic nucleus (DMH) has been proposed as a candidate for the neural substrate of a food-entrainable oscillator. The existence of a food-entrainable oscillator in the mammalian nervous system was inferred previously from restricted feeding-induced behavioral rhythmicity in rodents with suprachiasmatic nucleus lesions. In the present study, we have characterized the circadian rhythmicity of behavior in Wfs1-deficient mice during ad libitum and restricted feeding. Based on the expression of Wfs1 protein in the DMH it was hypothesized that Wfs1-deficient mice will display reduced or otherwise altered food anticipatory activity. Wfs1 immunoreactivity in DMH was found almost exclusively in the compact part. Restricted feeding induced c-Fos immunoreactivity primarily in the ventral and lateral aspects of DMH and it was similar in both genotypes. Wfs1-deficiency resulted in significantly lower body weight and reduced wheel-running activity. Circadian rhythmicity of behavior was normal in Wfs1-deficient mice under ad libitum feeding apart from elongated free-running period in constant light. The amount of food anticipatory activity induced by restricted feeding was not significantly different between the genotypes. Present results indicate that the effects of Wfs1-deficiency on behavioral rhythmicity are subtle suggesting that Wfs1 is not a major player in the neural networks responsible for circadian rhythmicity of behavior.

Altered pupillary light reflex in PACAP receptor 1-deficient mice.

The pupillary light reflex (PLR) is regulated by the classical photoreceptors, rods and cones, and by intrinsically photosensitive retinal ganglion cells (ipRGCs) expressing the photopigment melanopsin. IpRGCs receive input from rods and cones and project to the olivary pretectal nucleus (OPN), which is the primary visual center involved in PLR. Mice lacking either the classical photoreceptors or melanopsin exhibit some changes in PLR, whereas the reflex is completely lost in mice deficient of all three photoreceptors. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is co-stored with melanopsin in ipRGCs and mediates light signaling to the brain via the specific PACAP receptor 1 (PAC1R). Here, we examined the occurrence of PACAP and PAC1R in the mouse OPN, and studied if lack of PAC1R affected the PLR. PACAP-immunoreactive nerve fibers were shown in the mouse OPN, and by in situ hybridization histochemistry, we demonstrated the presence of PAC1R mRNA. Mice lacking PAC1R exhibited a significantly attenuated PLR compared to wild type mice upon light stimulation, and the difference became more pronounced as light intensity was increased. Our findings accord well with observations of the PLR in the melanopsin-deficient mouse. We conclude that PACAP/PAC1R signaling is involved in the sustained phase of the PLR at high irradiances.

Neuroglobin-deficiency exacerbates Hif1A and c-FOS response, but does not affect neuronal survival during severe hypoxia in vivo.

BACKGROUND: Neuroglobin (Ngb), a neuron-specific globin that binds oxygen in vitro, has been proposed to play a key role in neuronal survival following hypoxic and ischemic insults in the brain. Here we address whether Ngb is required for neuronal survival following acute and prolonged hypoxia in mice genetically Ngb-deficient (Ngb-null). Further, to evaluate whether the lack of Ngb has an effect on hypoxia-dependent gene regulation, we performed a transcriptome-wide analysis of differential gene expression using Affymetrix Mouse Gene 1.0 ST arrays. Differential expression was estimated by a novel data analysis approach, which applies non-parametric statistical inference directly to probe level measurements. PRINCIPAL FINDINGS: Ngb-null mice were born in expected ratios and were normal in overt appearance, home-cage behavior, reproduction and longevity. Ngb deficiency had no effect on the number of neurons, which stained positive for surrogate markers of endogenous Ngb-expressing neurons in the wild-type (wt) and Ngb-null mice after 48 hours hypoxia. However, an exacerbated hypoxia-dependent increase in the expression of c-FOS protein, an immediate early transcription factor reflecting neuronal activation, and increased expression of Hif1A mRNA were observed in Ngb-null mice. Large-scale gene expression analysis identified differential expression of the glycolytic pathway genes after acute hypoxia in Ngb-null mice, but not in the wts. Extensive hypoxia-dependent regulation of chromatin remodeling, mRNA processing and energy metabolism pathways was apparent in both genotypes. SIGNIFICANCE: According to these results, it appears unlikely that the loss of Ngb affects neuronal viability during hypoxia in vivo. Instead, Ngb-deficiency appears to enhance the hypoxia-dependent response of Hif1A and c-FOS protein while also altering the transcriptional regulation of the glycolytic pathway. Bioinformatic analysis of differential gene expression yielded novel predictions suggesting that chromatin remodeling and mRNA metabolism are among the key regulatory mechanisms when adapting to prolonged hypoxia.

The redundancy of recursion and infinity for natural language.

An influential line of thought claims that natural language and arithmetic processing require recursion, a putative hallmark of human cognitive processing (Chomsky in Evolution of human language: biolinguistic perspectives. Cambridge University Press, Cambridge, pp 45-61, 2010; Fitch et al. in Cognition 97(2):179-210, 2005; Hauser et al. in Science 298(5598):1569-1579, 2002). First, we question the need for recursion in human cognitive processing by arguing that a generally simpler and less resource demanding process--iteration--is sufficient to account for human natural language and arithmetic performance. We argue that the only motivation for recursion, the infinity in natural language and arithmetic competence, is equally approachable by iteration and recursion. Second, we submit that the infinity in natural language and arithmetic competence reduces to imagining infinite embedding or concatenation, which is completely independent from the ability to implement infinite processing, and thus, independent from both recursion and iteration. Furthermore, we claim that a property of natural language is physically uncountable finity and not discrete infinity.

Myg1-deficient mice display alterations in stress-induced responses and reduction of sex-dependent behavioural differences.

Myg1 (Melanocyte proliferating gene 1) is a highly conserved and ubiquitously expressed gene, which encodes a protein with mitochondrial and nuclear localization. In the current study we demonstrate a gradual decline of Myg1 expression during the postnatal development of the mouse brain that suggests relevance for Myg1 in developmental processes. To study the effects of Myg1 loss-of-function, we created Myg1-deficient (-/-) mice by displacing the entire coding sequence of the gene. Initial phenotyping, covering a multitude of behavioural, cognitive, neurological, physiological and stress-related responses, revealed that homozygous Myg1 (-/-) mice are vital, fertile and display no gross abnormalities. Myg1 (-/-) mice showed an inconsistent pattern of altered anxiety-like behaviour in different tests. The plus-maze and social interaction tests revealed that male Myg1 (-/-) mice were significantly less anxious than their wild-type littermates; female (-/-) mice showed increased anxiety in the locomotor activity arena. Restraint-stress significantly reduced the expression of the Myg1 gene in the prefrontal cortex of female wild-type mice and restrained female (-/-) mice showed a blunted corticosterone response, suggesting involvement of Myg1 in stress-induced responses. The main finding of the present study was that Myg1 invalidation decreases several behavioural differences between male and female animals that were obvious in wild-type mice, indicating that Myg1 contributes to the expression of sex-dependent behavioural differences in mice. Taken together, we provide evidence for the involvement of Myg1 in anxiety- and stress-related responses and suggest that Myg1 contributes to the expression of sex-dependent behavioural differences.

Relation between increased anxiety and reduced expression of alpha1 and alpha2 subunits of GABA(A) receptors in Wfs1-deficient mice.

Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. In clinical studies a relation between mutations in the Wfs1 gene and increased susceptibility for mood disorders has been established. According to our previous studies, mice lacking Wfs1 gene displayed increased anxiety in stressful environment. As the GABA-ergic system plays a significant role in the regulation of anxiety, we analyzed the expression of GABA-related genes in the forebrain structures of wild-type and Wfs1-deficient mice. Experimentally naïve Wfs1-deficient animals displayed a significant down-regulation of alpha1 (Gabra1) and alpha2 (Gabra2) subunits of GABA(A) receptors in the temporal lobe and frontal cortex. Exposure of wild-type mice to the elevated plus-maze decreased levels of Gabra1 and Gabra2 genes in the temporal lobe. A similar tendency was also established in the frontal cortex of wild-type animals exposed to behavioral test. In Wfs1-deficient mice the elevated plus-maze exposure did not induce further changes in the expression of Gabra1 and Gabra2 genes. By contrast, the expression of Gad1 and Gad2 genes, enzymes responsible for the synthesis of GABA, was not significantly affected by the exposure of mice to the elevated plus-maze or by the invalidation of Wfs1 gene. Altogether, the present study demonstrates that increased anxiety of Wfs1-deficient mice is probably linked to reduced expression of Gabra1 and Gabra2 genes in the frontal cortex and temporal lobe.

Wfs1-deficient mice display impaired behavioural adaptation in stressful environment.

Wfs1-deficient mice were generated by disrupting the 8th exon of Wfs1 gene. Reproduction rates of homozygous Wfs1-deficient mice were slightly below the expected values, they displayed intolerance to glucose and overall lower body weight. The present behavioural study was performed in female Wfs1-deficient mice due to their milder metabolic disturbances. Non-fasting blood glucose levels did not differ between homozygous Wfs1-deficient mice and wild-type littermates. While there was no difference in baseline plasma corticosterone, exposure to stress induced a nearly three-fold elevation of corticosterone in Wfs1-deficient mice in relation to wild-type littermates. Wfs1-deficient mice did not display obvious shortcomings in sensory and motor functioning as exemplified by intact responses in conditioned learning paradigms and rota-rod test. Locomotor activity of Wfs1-deficient mice was significantly lower only in brightly lit environment. Short-term isolation had a significant anxiogenic-like effect on the behaviour of Wfs1-deficient mice in dark/light exploration test. Lower exploratory activity of Wfs1-deficient mice in the plus-maze was antagonised by pre-treatment with diazepam (1 mg/kg), a GABA(A) receptor agonist. Wfs1-deficient mice displayed increased anxiety-like behaviour in hyponeophagia test. The locomotor stimulatory effects of amphetamine (2.5-7.5 mg/kg) and apomorphine (3 mg/kg) were significantly attenuated and facilitated, respectively, in Wfs1-deficient mice. There were no differences between Wfs1-deficient mice and wild-types in forced swimming behaviour and conditioned fear responses. Subtle impairments in reversal learning were apparent in Wfs1-deficient mice in the Morris water maze. Altogether, the present study demonstrates impaired behavioural adaptation of Wfs1-deficient mice in stress-inducing situations. It is likely that Wfs1 protein plays a major role in the behavioural adaptation mechanisms to novel and stressful environments.

Characterization of MYG1 gene and protein: subcellular distribution and function.

BACKGROUND INFORMATION: MYG1 [Melanocyte proliferating gene 1, also known as Gamm1 (NM_021640)] is a recently described gene of unknown function. MYG1 orthologues are found in simple as well as complex eukaryotes. According to sequence homology, MYG1 is considered to have a metal-dependent protein hydrolase (UPF0160) domain. The purpose of the present study was to determine the expression and subcellular localization of MYG1 protein and to identify physiological processes connected to MYG1 function. RESULTS: Human and mouse MYG1 is ubiquitously expressed, with the highest level in the testis. Analysis of mouse embryos moreover revealed a uniform Myg1 expression at E (embryonic day) 8.5, but at E11.75 expression becomes restricted predominantly to the developing brain and eye, limb buds and tail region. MYG1 exhibits a mitochondrial targeting signal in the N-terminal region and a Pat7-type nuclear localization signal in the region between amino acids 33-39 and localizes to these compartments. No active shuttling of MYG1 between the nucleus and the mitochondria was detected and the distribution of MYG1 was not dependent on the phase of the cell cycle. Immunoprecipitation of C-terminally FLAG-tagged MYG1 from HeLa cells did not identify any co-precipitated proteins. siRNA (short interfering RNA)-mediated knockdown of MYG1 mRNA was mainly followed by changes in the level of transcripts encoding factors involved in developmental tissue patterning and growth as well as immune-related processes. CONCLUSIONS: Taken together, we infer that MYG1 is a ubiquitous nucleo-mitochondrial protein, with differential pattern and level of expression during embryonic development. MYG1 expression in normal adult tissues is stable and our data suggest MYG1 involvement in early developmental processes and also in adult stress/illness conditions.