Riin Reimets

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.

Effect of permanent middle cerebral artery occlusion on Cytoglobin expression in the mouse brain.

Cytoglobin, a new member of the mammalian heme-globin family has been shown to bind oxygen and to have cell protective properties in vitro. Cytoglobin is specifically expressed in a subpopulation of brain neurons. Based on hypoxia-induced up regulation and proposed scavenging of reactive oxygen species Cytoglobin was suggested as a candidate for pharmaceutical stroke treatment. Since production of reactive oxygen species is a hallmark of ischemia, we hypothesized that Cytoglobin expression would be increased and that Cytoglobin expressing neurons would be spared after ischemic injury. Twenty male C57BL/6J mice were used in the experimental design. Ten were sham operated and ten were given permanent middle cerebral artery occlusion (pMCAo). All animals were euthanized after 24h. From each group, three animals were used for histology and seven for QRT-PCR and western blotting. Immunohistochemical examination of the ischemic penumbra revealed neither changes in Cytoglobin immunoreactivity nor any changes in expression in the necrotic infarct area. The lack of expression change was confirmed by western blotting and QRT-PCR showing no significant difference between sham and pMCAo operated mice. This suggests that Cytoglobin is likely not important for global neuronal protection following ischemia and the role of Cytoglobin in relation to endogenous neuroprotection remains unresolved.

Gene expression patterns and environmental enrichment-induced effects in the hippocampi of mice suggest importance of Lsamp in plasticity

Limbic system associated membrane protein (Lsamp) gene is involved in behavioral adaptation in social and anxiogenic environments and has been associated with a broad spectrum of psychiatric diseases. Here we studied the activity of alternative promoters of Lsamp gene in mice in three rearing conditions (standard housing, environmental enrichment and social isolation) and in two different genetic backgrounds (129S6/SvEv and C57BL/6). Isolation had no effect on the expression levels of Lsamp. Environmental enrichment elevated the expression levels of Lsamp 1b transcript specifically in the hippocampus in B6 mice, and the same tendency existed across both mouse lines and both transcripts. Furthermore, we showed that the density of cells exhibiting 1b promoter activity is remarkably higher in the subgranular zone of the dentate gyrus in the hippocampal formation which is a specific area of enrichment-induced neurogenesis in adult rodents. On the contrary to 1b, 1a promoter is selectively active in the pyramidal and granule cell layers. We provide evidence that Lsamp modulates enrichment-induced activation of Bdnf as the enrichment-induced elevation of Bdnf in the hippocampus is significantly diminished in Lsamp-deficient mice; furthermore, a significant correlation was found between the expression levels of Lsamp and Bdnf transcripts in the hippocampus and frontal cortex. Significant strain differences in Lsamp expression were detected in the hippocampus, frontal cortex and thalamus that could be related to the different behavioral phenotype of B6 and 129Sv mice. Our data provides further evidence that LSAMP is implicated in the hippocampal connectivity and plasticity thereby modulating adaptability in changing environments.

Wfs1-deficient animals have brain-region-specific changes of Na+, K+-ATPase activity and mRNA expression of α1 and β1 subunits

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile‐onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic β‐cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1‐deficient mice have brain‐region‐specific changes in Na+,K+‐ATPase activity and in the expression of the α1 and β1 subunits. We found a significant (1.6‐fold) increase of Na‐pump activity and β1 subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild‐type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na‐pump activity 1.3‐fold in the midbrain and dorsal striatum and 2.0‐fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na‐pump α1‐subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1‐deficient homozygous animals compared with wild‐type littermates. In the temporal lobe, an increase in the activity of the Na‐pump is probably related to increased anxiety established in Wfs1‐deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1‐deficient mice may be associated with a decrease of Na‐pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM.

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.

Wfs1- deficient rats develop primary symptoms of Wolfram syndrome: insulin-dependent diabetes, optic nerve atrophy and medullary degeneration

Wolfram syndrome (WS) is a rare autosomal-recessive disorder that is caused by mutations in the WFS1 gene and is characterized by juvenile-onset diabetes, optic atrophy, hearing loss and a number of other complications. Here, we describe the creation and phenotype of Wfs1 mutant rats, in which exon 5 of the Wfs1 gene is deleted, resulting in a loss of 27 amino acids from the WFS1 protein sequence. These Wfs1-ex5-KO232 rats show progressive glucose intolerance, which culminates in the development of diabetes mellitus, glycosuria, hyperglycaemia and severe body weight loss by 12 months of age. Beta cell mass is reduced in older mutant rats, which is accompanied by decreased glucose-stimulated insulin secretion from 3 months of age. Medullary volume is decreased in older Wfs1-ex5-KO232 rats, with the largest decreases at the level of the inferior olive. Finally, older Wfs1-ex5-KO232 rats show retinal gliosis and optic nerve atrophy at 15 months of age. Electron microscopy revealed axonal degeneration and disorganization of the myelin in the optic nerves of older Wfs1-ex5-KO232 rats. The phenotype of Wfs1-ex5-KO232 rats indicates that they have the core symptoms of WS. Therefore, we present a novel rat model of WS.

Variability in the effect of antidepressants upon Wfs1-deficient mice is dependent on the drugs' mechanism of actions.

There is significant comorbidity between mood disorders and diabetes. Wolfram syndrome-related to deficient WFS1 gene function-causes diabetes and mood disorders in humans. Mice lacking the Wfs1 gene display impaired emotional behaviour and glucose metabolism. Various antidepressant drugs are used for alleviating the symptoms of mood disorders. For this study the tail suspension test and locomotor activity test were used to compare the effects of different antidepressants upon homozygous Wfs1-deficient, heterozygous Wfs1-deficient and wild-type mice. Mouse glucose metabolism was concurrently studied using the glucose tolerance test. We showed that ketamine(10mg/kg),NMDA antagonist, escitalopram(2.5-10mg/kg), selective serotonin reuptake inhibitor(SSRI), and amitriptyline(10mg/kg), noradrenaline and serotonin reuptake inhibitor, elicited a stronger antidepressant-like effect in homozygous Wfs1-deficient mice compared to wild-type mice. The effect of noradrenaline and serotonin reuptake inhibitor desipramine(10 and 20mg/kg) did not differ between genotypes. The dopamine and noradrenaline reuptake inhibitor bupropion(5-20mg/kg) had no significant antidepressant-like effect upon any genotype. Amitriptyline and desipramine potentiated a glucose elevation, escitalopram and bupropion did not affect glucose concentrations, and ketamine improved impaired glucose metabolism in homozygous Wfs1-deficient mice. Therefore, the results of this study suggest that SSRIs are the drugs of choice for the treatment of depressive symptoms in diabetic patients. The efficacy of ketamine for these patients remains to be established. Nonetheless, employing the mechanism of action of ketamine that affected glucose metabolism positively, could be an approach for development of improved antidepressants. Wfs1-deficient mice are likely the good animal model to develop new antidepressants more suitable for depressed patients with 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.

Hypothermia augments stress response in mammalian cells

ABSTRACT 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 HIF1A affecting the antioxidant system and hypoxia response pathways, respectively. At the same time, NF‐&kgr;B, 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. HIGHLIGHTSHypothermia extensively remodels gene expression.Mild hypothermia activates Nrf2 and HIF1 transcription factors.Hypothermic pretreatment protects against oxidative stress.Therapeutic effect of hypothermia stems from inhibitory and activating processes.

Muscarinic agonist ameliorates insulin secretion in Wfs1 deficient mouse

OBJECTIVES Similar to patients with Wolfram syndrome and to heterozygous Wolframin1 (Wfs1) mutation carriers, Wfs1-deficient mice exhibit impaired glucose tolerance and lower plasma insulin levels. Muscarinic receptor 3 agonists have previously been shown to potentiate glucose-stimulated insulin secretion. Therefore, the aim of this study was to investigate insulin-secretion dynamics in Wfs1-deficient mice and evaluate carbachol, muscarinic agonist and the ability to ameliorate the insulin secretion deficits caused by the Wfs1 mutation. METHODS Wild-type Wfs1 heterozygous and Wfs1 mutant mice were used. Blood glucose was measured after glucose and carbachol administration. Insulin secretion was measured from serum using ELISA. RESULTS Glucose administration causes hyperglycemia in Wfs1-deficient mice due to decreased insulin secretion. This deficit is abolished by administration of the muscarinic agonist carbachol. CONCLUSIONS Activation of the muscarinic pathway to potentiate insulin secretion may present a target to manage diabetes resulting from Wfs1 deficiency.