Anton Terasmaa

Effect of denervation of the locus coeruleus projections by DSP-4 treatment on [3H]-raclopride binding to dopamine D2 receptors and D2 receptor–G protein interaction in the rat striatum

Changes in the control of dopaminergic neurotransmission by noradrenergic locus coeruleus (LC) projections has been implicated in such disorders as depression, drug addiction, and Parkinsons disease. In the present study, the effect of DSP-4, a neurotoxin highly selective for LC projections, on D(2) receptor abundance as assessed by [3H]-raclopride binding in the striatum was studied in rats after administration in doses of 10 and 50 mg/kg either 3 days or 1 month before decapitation. Three days after DSP-4 the levels of noradrenaline in the frontal cortex were dose-dependently reduced; after 1 month, noradrenaline levels were lowered only by the higher dose. DOPAC levels were dose-dependently reduced in the frontal cortex and striatum 3 days but not 1 month after DSP-4 treatment. Cortical 5-HIAA levels were reduced 3 days but not 1 month after DSP-4. The apparent number of D(2) receptor binding sites in the striatum was higher 1 month after either dose of DSP-4. DSP-4 treatment had no effect on [3H]-raclopride binding affinity, the ability of dopamine (DA) to compete with [3H]-raclopride binding and to activate [35S]GTPgammaS binding or on the binding affinities of GDP and [35S]GTPgammaS for corresponding G proteins 1 month after administration of the neurotoxin. These data suggest that after administration of DSP-4, short-term reduction in DA and 5-HT metabolism occurs. Subsequently, an upregulation of D(2) receptor binding sites develops in the striatum even after a minor denervation of the LC projections. Thus, alterations in the LC projection systems elicit lasting adaptive changes in DA-ergic neurotransmission that can serve as a substrate for psychiatric disorders.

Wfs1 gene deletion causes growth retardation in mice and interferes with the growth hormone pathway

The aim of present study was to describe changes in gene expression in the temporal lobe of mice induced by deletion of the Wfs1 gene. Temporal lobes samples were analyzed using Affymetrix Mouse Genome 420 2 GeneChips and expression profiles were functionally annotated with GSEA and Ingenuity Pathway Analysis. We found that Wfs1 mutant mice are significantly smaller (20.9 +/- 1.6 g) than their wild-type counterparts (31.0 +/- 0.6 g, P < 0.0001). This difference existed in 129S6 and C57B6 backgrounds. Interestingly, microarray analysis identified upregulation of growth hormone (GH) transcripts and functional analysis revealed activation of GH pathways. In line with microarray data, the level of IGF-1 in the plasma of Wfs1 mutant mice was significantly increased (P < 0.05). Thus, Wfs1 deletion induces growth retardation, whereas the GH pathway is activated. To test the interaction between the Wfs1 deletion and genomic background, mutant mice were backcrossed to two different genetic backgrounds. In line with previous studies, an interaction between a gene knockout and genetic background was found in gene expression profiles in the congenic region. However, genetic background did not alter the effect of the Wfs1 mutation on either body weight or GH pathway activation. Further studies are needed to describe biochemical and molecular changes of the growth hormone axis as well as in other hormones to clarify their role in growth retardation in the Wfs1 mutant mice.

Wfs1 mutation makes mice sensitive to insulin-like effect of acute valproic acid and resistant to streptozocin.

Valproic acid (VLP) is a widely used anticonvulsant and mood-stabilizing drug that relieves the endoplasmic reticulum (ER) stress response, a pathogenetic process related to diabetes. The aim of the present study was to evaluate whether acute valproic acid is able to interfere with glucose intolerance in two different diabetes models: The first model was a Wfs1 mutant mouse with an elevated ER stress response and the second model a streptozocin-induced diabetic mouse. VLP (300xa0mg/kg, i.p.) was administered to Wfs1 knockout (KO) mice and glucose tolerance test was performed 15xa0min later. VLP did not have an effect on the course of the glucose tolerance test in wild-type mice, while it did normalize the glucose intolerance in Wfs1 knockout mice. Acute valproic acid also lowered the blood glucose levels in streptozocin-treated mice and potentiated the effect of insulin in these mice. Thus, acute valproic acid is effective in lowering blood glucose levels possibly by potentiating insulin action in both Wfs1 KO mice and in streptozocin-induced type 1 diabetic mice.

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.

Impaired striatal dopamine output of homozygous Wfs1 mutant mice in response to [K+] challenge

Loss of function of the Wfs1 gene causes Wolfram syndrome, a rare multisystem degenerative disorder. Mutant mice with targeted Wfs1 gene disruption (Wfs1 KO) display morphological and behavioral impairments that are not well understood. The present study aimed to investigate the striatal dopamine output of wild-type, heterozygous, and homozygous Wfs1 null-mutant mice using in vivo microdialysis technique. The baseline dopamine output in striatum was similar in all three animal groups. The application of 100xa0mM [K+]-rich modified Ringer solution caused in homozygous Wfs1 mutant mice an increase of dopamine output by 400%, while in wild-type and heterozygous animals, the increase of the dopamine output yielded up to 1,200%. In sum, the homozygous Wfs1 mutant mice (AUC0–3 = 0.212xa0nM/μlxa0h) show significantly decreased striatal dopamine output in response to high-concentration [K+] challenge as compared with wild-type or heterozygous Wfs1 mutant conspecifics (AUC0–3u2009=u20090.427 and 0.505xa0nM/μlxa0h, respectively). This could explain at least some of the behavioral alterations in Wfs1 mutant mice.

Effect of chronic valproic acid treatment on hepatic gene expression profile in wfs1 knockout mouse

Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300u2009mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0u2009ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype.

Hypothalamic gene expression profile indicates a reduction in G protein signaling in the Wfs1 mutant mice

The Wfs1 gene codes for a protein with unknown function, but deficiency in this protein results in a range of neuropsychiatric and neuroendocrine syndromes. In the present study we aimed to find the functional networks influenced by Wfs1 in the hypothalamus. We performed gene expression profiling (Mouse Gene 1.0 ST Arrays) in Wfs1-deficient mice; 305 genes were differentially expressed with nominal P value<0.01. FDR (false discovery rate)-adjusted P values were significant (0.007) only for two genes: C4b (t=9.66) and Wfs1 (t=-9.03). However, several genes related to G protein signaling were very close to the FDR-adjusted significance level, such as Rgs4 (regulator of G protein signaling 4) that was downregulated (-0.34, t=-5.4) in Wfs1-deficient mice. Changes in Rgs4 and C4b expression were confirmed by QRT-PCR. In humans, Rgs4 is in the locus for bipolar disease (BPD), and its expression is downregulated in BPD. C4b is a gene related to the neurodegenerative diseases. Functional analysis including the entire data set revealed significant alterations in the canonical pathway G protein-coupled receptor signaling. The gene expression profile in the hypothalami of the Wfs1 mutant mice was significantly similar to the profiles of following biological functions: psychological disorders, bipolar disorder, mood disorder. In conclusion, hypothalamic gene expression profile resembles with some molecular pathways functionally related to the clinical syndromes in the Wolfram syndrome patients.

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.

Prohormone convertase 2 activity is increased in the hippocampus of Wfs1 knockout mice.

Background: Mutations in WFS1 gene cause Wolfram syndrome, which is a rare autosomal recessive disorder, characterized by diabetes insipidus, diabetes mellitus, optic nerve atrophy, and deafness. The WFS1 gene product wolframin is located in the endoplasmic reticulum. Mice lacking this gene exhibit disturbances in the processing and secretion of peptides, such as vasopressin and insulin. In the brain, high levels of the wolframin protein have been observed in the hippocampus, amygdala, and limbic structures. The aim of this study was to investigate the effect of Wfs1 knockout (KO) on peptide processing in mouse hippocampus. A peptidomic approach was used to characterize individual peptides in the hippocampus of wild-type and Wfs1 KO mice. Results: We identified 126 peptides in hippocampal extracts and the levels of 10 peptides differed between Wfs1 KO and wild-type mice at P < 0.05. The peptide with the largest alteration was little-LEN, which level was 25 times higher in the hippocampus of Wfs1 KO mice compared to wild-type mice. Processing (cleavage) of little-LEN from the Pcsk1n gene product proSAAS involves prohormone convertase 2 (PC2). Thus, PC2 activity was measured in extracts prepared from the hippocampus of Wfs1 KO mice. The activity of PC2 in Wfs1 mutant mice was significantly higher (149.9 ± 2.3%, p < 0.0001, n = 8) than in wild-type mice (100.0 ± 7.0%, n = 8). However, Western blot analysis showed that protein levels of 7B2, proPC2 and PC2 were same in both groups, and so were gene expression levels. Conclusion: Processing of proSAAS is altered in the hippocampus of Wfs1-KO mice, which is caused by increased activity of PC2. Increased activity of PC2 in Wfs1 KO mice is not caused by alteration in the levels of PC2 protein. Our results suggest a functional link between Wfs1 and PC2. Thus, the detailed molecular mechanism of the role of Wfs1 in the regulation of PC2 activity needs further investigation.

Preventive treatment with liraglutide protects against development of glucose intolerance in a rat model of Wolfram syndrome

Wolfram syndrome (WS) is a rare autosomal recessive disorder caused by mutations in the WFS1 (Wolframin1) gene. The syndrome first manifests as diabetes mellitus, followed by optic nerve atrophy, deafness, and neurodegeneration. The underlying mechanism is believed to be a dysregulation of endoplasmic reticulum (ER) stress response, which ultimately leads to cellular death. Treatment with glucagon-like peptide-1 (GLP-1) receptor agonists has been shown to normalize ER stress response in several in vitro and in vivo models. Early chronic intervention with the GLP-1 receptor agonist liraglutide starting before the onset of metabolic symptoms prevented the development of glucose intolerance, improved insulin and glucagon secretion control, reduced ER stress and inflammation in Langerhans islets in Wfs1 mutant rats. Thus, treatment with GLP-1 receptor agonists might be a promising strategy as a preventive treatment for human WS patients.