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.

Chronic Stress Alters Hippocampal Renin-Angiotensin-Aldosterone System Component Expression in an Aged Rat Model of Wolfram Syndrome.

Biallelic mutations in the gene encoding WFS1 underlie the development of Wolfram syndrome (WS), a rare neurodegenerative disorder with no available cure. We have previously shown that Wfs1 deficiency can impair the functioning of the renin-angiotensin-aldosterone system (RAAS). The expression of two key receptors, angiotensin II receptor type 2 (Agtr2) and bradykinin receptor B1 (Bdkrb1), was downregulated both in vitro and in vivo across multiple organs in a rat model of WS. Here, we show that the expression of key RAAS components is also dysregulated in neural tissue from aged WS rats and that these alterations are not normalized by pharmacological treatments (liraglutide (LIR), 7,8-dihydroxyflavone (7,8-DHF) or their combination). We found that the expression of angiotensin II receptor type 1a (Agtr1a), angiotensin II receptor type 1b (Agtr1b), Agtr2 and Bdkrb1 was significantly downregulated in the hippocampus of WS animals that experienced chronic experimental stress. Treatment-naïve WS rats displayed different gene expression patterns, underscoring the effect of prolonged experiment-induced stress. Altogether, we posit that Wfs1 deficiency disturbs RAAS functioning under chronic stressful conditions, thereby exacerbating neurodegeneration in WS.

Treatment with the dual-incretin agonist DA-CH5 demonstrates potent therapeutic effect in a rat model of Wolfram Syndrome.

Aim: Wolfram Syndrome (WS) is a rare condition caused by mutations in Wfs1, with a poor prognosis and no cure. Mono-agonists targeting the incretin glucagon-like-peptide 1 (GLP-1) have demonstrated disease-modifying potential in pre-clinical and clinical settings. Dual agonists that target GLP-1 and glucose-dependent insulinotropic polypeptide (GIP-1) are reportedly more efficacious; hence, we evaluated the therapeutic potential of dual incretin agonism in a loss-of-function rat model of WS. Methods: Eight-month-old Wfs1 knock-out (KO) and wild-type control rats were continuously treated with either the dual agonist DA-CH5 or saline for four months. Glycemic profile, visual acuity and hearing sensitivity were longitudinally monitored pre-treatment, and then at 10.5 and 12 months. Pancreata and retina were harvested for immunohistological analysis. Results: DA-CH5 therapy reversed glucose intolerance in KO rats and provided lasting anti-diabetogenic protection. Treatment also reversed intra-islet alterations, including reduced endocrine islet area and ß-cell density, indicating its regenerative potential. Although no rescue effect was noted for hearing loss, visual acuity and retinal ganglion cell density were better preserved in DA-CH5-treated rats. Conclusion: We present preclinical evidence for the pleiotropic therapeutic effects of long-term dual incretin agonist treatment; effects were seen despite treatment beginning after symptom-onset, indicating reversal of disease progression. Dual incretins represent a promising therapeutic avenue for WS patients.

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.

Liraglutide, 7,8-DHF and their co-treatment prevents loss of vision and cognitive decline in a Wolfram syndrome rat model.

Wolfram syndrome (WS) is a monogenic progressive neurodegenerative disease and is characterized by various neurological symptoms, such as optic nerve atrophy, loss of vision, cognitive decline, memory impairment, and learning difficulties. GLP1 receptor agonist liraglutide and BDNF mimetic 7,8-dihydroxyflavone (7,8-DHF) have had protective effect to visual pathway and to learning and memory in different rat models of neurodegenerative disorders. Although synergistic co-treatment effect has not been reported before and therefore the aim of the current study was to investigate liraglutide, 7,8-DHF and most importantly for the first time their co-treatment effect on degenerative processes in WS rat model. We took 9 months old WS rats and their wild-type (WT) control animals and treated them daily with liraglutide, 7,8-DHF or with the combination of liraglutide and 7,8-DHF up to the age of 12.5 months (n = 47, 5-8 per group). We found that liraglutide, 7,8-DHF and their co-treatment all prevented lateral ventricle enlargement, improved learning in Morris Water maze, reduced neuronal inflammation, delayed the progression of optic nerve atrophy, had remyelinating effect on optic nerve and thereby improved visual acuity in WS rats compared to WT controls. Thus, the use of the liraglutide, 7,8-DHF and their co-treatment could potentially be used as a therapeutic intervention to induce neuroprotection or even neuronal regeneration.

Early Intervention and Lifelong Treatment with GLP1 Receptor Agonist Liraglutide in a Wolfram Syndrome Rat Model with an Emphasis on Visual Neurodegeneration, Sensorineural Hearing Loss and Diabetic Phenotype.

Wolfram syndrome (WS), also known as a DIDMOAD (diabetes insipidus, early-onset diabetes mellitus, optic nerve atrophy and deafness) is a rare autosomal disorder caused by mutations in the Wolframin1 (WFS1) gene. Previous studies have revealed that glucagon-like peptide-1 receptor agonist (GLP1 RA) are effective in delaying and restoring blood glucose control in WS animal models and patients. The GLP1 RA liraglutide has also been shown to have neuroprotective properties in aged WS rats. WS is an early-onset, chronic condition. Therefore, early diagnosis and lifelong pharmacological treatment is the best solution to control disease progression. Hence, the aim of this study was to evaluate the efficacy of the long-term liraglutide treatment on the progression of WS symptoms. For this purpose, 2-month-old WS rats were treated with liraglutide up to the age of 18 months and changes in diabetes markers, visual acuity, and hearing sensitivity were monitored over the course of the treatment period. We found that treatment with liraglutide delayed the onset of diabetes and protected against vision loss in a rat model of WS. Therefore, early diagnosis and prophylactic treatment with the liraglutide may also prove to be a promising treatment option for WS patients by increasing the quality of life.

High-Fat Diet Induces Pre-Diabetes and Distinct Sex-Specific Metabolic Alterations in Negr1-Deficient Mice.

In the large GWAS studies, NEGR1 gene has been one of the most significant gene loci for body mass phenotype. The purpose of the current study was to clarify the role of NEGR1 in the maintenance of systemic metabolism, including glucose homeostasis, by using both male and female Negr1-/- mice receiving a standard or high fat diet (HFD). We found that 6 weeks of HFD leads to higher levels of blood glucose in Negr1-/- mice. In the glucose tolerance test, HFD induced phenotype difference only in male mice; Negr1-/- male mice displayed altered glucose tolerance, accompanied with upregulation of circulatory branched-chain amino acids (BCAA). The general metabolomic profile indicates that Negr1-/- mice are biased towards glyconeogenesis, fatty acid synthesis, and higher protein catabolism, all of which are amplified by HFD. Negr1 deficiency appears to induce alterations in the efficiency of energy storage; reduced food intake could be an attempt to compensate for the metabolic challenge present in the Negr1-/- males, particularly during the HFD exposure. Our results suggest that the presence of functional Negr1 allows male mice to consume more HFD and prevents the development of glucose intolerance, liver steatosis, and excessive weight gain.

The Expression of RAAS Key Receptors, Agtr2 and Bdkrb1, Is Downregulated at an Early Stage in a Rat Model of Wolfram Syndrome.

Wolfram syndrome (WS) 1 is a rare monogenic neurodegenerative disorder caused by mutations in the gene encoding WFS1. Knowledge of the pathophysiology of WS is incomplete and to date, there is no treatment available. Here, we describe early deviations in the renin-angiotensin-aldosterone system (RAAS) and bradykinin pathway (kallikrein kinin system, KKS) observed in a rat model of WS (Wfs1 KO) and the modulative effect of glucagon-like peptide-1 receptor agonist liraglutide (LIR) and anti-epileptic drug valproate (VPA), which have been proven effective in delaying WS progression in WS animal models. We found that the expression of key receptors of the RAAS and KKS, Agtr2 and Bdkrb1, were drastically downregulated both in vitro and in vivo at an early stage in a rat model of WS. Moreover, in Wfs1, KO serum aldosterone levels were substantially decreased and bradykinin levels increased compared to WT animals. Neither treatment nor their combination affected the gene expression levels seen in the Wfs1 KO animals. However, all the treatments elevated serum aldosterone and decreased bradykinin in the Wfs1 KO rats, as well as increasing angiotensin II levels independent of genotype. Altogether, our results indicate that Wfs1 deficiency might disturb the normal functioning of RAAS and KKS and that LIR and VPA have the ability to modulate these systems.

Muscarinic Agonist Ameliorates Insulin Secretion in Wfs1-Deficient Mice.

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.

GLP-1 receptor agonist liraglutide has a neuroprotective effect on an aged rat model of Wolfram syndrome.

Wolfram syndrome (WS) is a rare neurodegenerative disorder that is mainly characterized by diabetes mellitus, optic nerve atrophy, deafness, and progressive brainstem degeneration. Treatment with GLP-1 receptor agonists has shown a promising anti-diabetic effect in WS treatment in both animal models and in human patients. Since previous research has tended to focus on investigation of the WS first symptom, diabetes mellitus, the aim of the present study was to examine liraglutide effect on WS-associated neurodegeneration. We took 9-month-old Wfs1 knock-out (KO) animals that already had developed glucose intolerance and treated them with liraglutide for 6 months. Our research results indicate that 6-month liraglutide treatment reduced neuroinflammation and ameliorated endoplasmic reticulum (ER) stress in the inferior olive of the aged WS rat model. Liraglutide treatment also protected retinal ganglion cells from cell death and optic nerve axons from degeneration. According to this, the results of the present study provide novel insight that GLP-1 receptor agonist liraglutide has a neuroprotective effect in the WS rat model.

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.

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.

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.

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.

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.

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.