Major depression is a highly prevalent severe mood disorder that is treated with antidepressants. The molecular targets of antidepressants require definition. We investigated the role of the acid sphingomyelinase (Asm)-ceramide system as a target for antidepressants. Therapeutic concentrations of the antidepressants amitriptyline and fluoxetine reduced Asm activity and ceramide concentrations in the hippocampus, increased neuronal proliferation, maturation and survival and improved behavior in mouse models of stress-induced depression. Genetic Asm deficiency abrogated these effects. Mice overexpressing Asm, heterozygous for acid ceramidase, treated with blockers of ceramide metabolism or directly injected with C16 ceramide in the hippocampus had higher ceramide concentrations and lower rates of neuronal proliferation, maturation and survival compared with controls and showed depression-like behavior even in the absence of stress. The decrease of ceramide abundance achieved by antidepressant-mediated inhibition of Asm normalized these effects. Lowering ceramide abundance may thus be a central goal for the future development of antidepressants. DOI: https://doi.org/10. 1038/nm.3214 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-79905 Accepted Version Originally published at: Gulbins, E; Palmada, M; Reichel, M; Lüth, A; Böhmer, C; Amato, D; Müller, C P; Tischbirek, C H; Groemer, T W; Tabatabai, G; Becker, K A; Tripal, P; Staedtler, S; Ackermann, T F; van Brederode, J; Alzheimer, C; Weller, M; Lang, U E; Kleuser, B; Grassme, H; Kornhuber, J (2013). Acid sphingomyelinaseceramide system mediates effects of antidepressant drugs. Nature Medicine, 19 (7) Major depression may be triggered by psychological stress, inflammatory cytokines, and dysfunction of the hypothalamic-pituitary-adrenal axis, etc. 1-4 .The previously held monoamine hypothesis for the action of antidepressants has been questioned because the antidepressant effect of these drugs is not clearly associated with their monoaminergic effect; in fact, the antidepressant tianeptine is even a serotonin reuptake enhancer 5 . Furthermore, the direct effect on monoamines contrasts with the delay of antidepressant effects in patients. Recent concepts of the pathogenesis of major depression suggest a change of cellular plasticity predominantly in the hippocampus and a shift in the balance between neurogenic and antiapoptotic events that leads to neurodegeneration and hippocampal atrophy [6][7][8][9] . Antidepressants increase neurogenesis and reverse hippocampal atrophy associated with major depression 9 .Here, we tested the role of the acid sphingomyelinase (EC 3.1.4.12, sphingomyelin phosphodiesterase, human protein: ASM, murine protein: Asm, gene symbol: Smpd1) and ceramide system as a target for antidepressants. Asm is ubiquitously expressed and releases ceramide from sphingomyelin, predominantly in lysosomes but also in secretory lysosomes and on the plasma membrane 10-13 .The antidepres...

Klotho is a membrane protein participating in the inhibitory effect of FGF23 on the formation of 1,25-dihydroxyvitamin-D(3) [1,25(OH)(2)D(3)]. It participates in the regulation of renal tubular phosphate reabsorption and stimulates renal tubular Ca(2+) reabsorption. Klotho hypomorphic mice (klotho(hm)) suffer from severe growth deficit, rapid aging, and early death, events largely reversed by a vitamin D-deficient diet. The present study explored the role of Klotho deficiency in mineral and electrolyte metabolism. To this end, klotho(hm) mice and wild-type mice (klotho(+/+)) were subjected to a normal (D(+)) or vitamin D-deficient (D(-)) diet or to a vitamin D-deficient diet for 4 wk and then to a normal diet (D(-/+)). At the age of 8 wk, body weight was significantly lower in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice, klotho(hm)D(-) mice, and klotho(hm)D(-/+) mice. Plasma concentrations of 1,25(OH)(2)D(3,) adrenocorticotropic hormone (ACTH), antidiuretic hormone (ADH), and aldosterone were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. Plasma volume was significantly smaller in klotho(hm)D(-/+) mice, and plasma urea, Ca(2+), phosphate and Na(+), but not K(+) concentrations were significantly higher in klotho(hm)D(+) mice than in klotho(+/+)D(+) mice. The differences were partially abrogated by a vitamin D-deficient diet. Moreover, the hyperaldosteronism was partially reversed by Ca(2+)-deficient diet. Ussing chamber experiments revealed a marked increase in amiloride-sensitive current across the colonic epithelium, pointing to enhanced epithelial sodium channel (ENaC) activity. A salt-deficient diet tended to decrease and a salt-rich diet significantly increased the life span of klotho(hm)D(+) mice. In conclusion, the present observation disclose that the excessive formation of 1,25(OH)(2)D(3) in Klotho-deficient mice results in extracellular volume depletion, which significantly contributes to the shortening of life span.

The serum- and glucocorticoid-inducible kinase 1 (SGK1) is transcriptionally upregulated by mineralocorticoids and activated by insulin. The kinase enhances renal tubular Na+-reabsorption and accounts for blood pressure increase following high salt diet in mice made hyperinsulinemic by dietary fructose or fat. The present study describes the in vitro and in vivo efficacy of a novel SGK1 inhibitor (EMD638683). EMD638683 was tested in vitro by determination of SGK1-dependent phosphorylation of NDRG1 (N-Myc downstream-regulated gene 1) in human cervical carcinoma HeLa-cells. In vivo EMD638683 (4460 ppm in chow, i.e. approx. 600 mg/kg/day) was administered to mice drinking tap water or isotonic saline containing 10% fructose. Blood pressure was determined by the tail cuff method, and urinary electrolyte (flame photometry) concentrations determined in metabolic cages. In vitro testing disclosed EMD638683 as a SGK1 inhibitor with an IC50 of 3 µM. Within 24 hours in vivo EMD638683 treatment significantly decreased blood pressure in fructose/saline-treated mice but not in control animals or in SGK1 knockout mice. EMD638683 failed to alter the blood pressure in SGK1 knockout mice. Following chronic (4 weeks) fructose/high salt treatment, additional EMD638683 treatment again decreased blood pressure. EMD638683 thus abrogates the salt sensitivity of blood pressure in hyperinsulinism without appreciably affecting blood pressure in the absence of hyperinsulinism. EMD638683 tended to increase fluid intake and urinary excretion of Na⁺, significantly increased urinary flow rate and significantly decreased body weight. Conclusion: EMD638683 could serve as a template for drugs counteracting hypertension in individuals with type II diabetes and metabolic syndrome.

The pore-forming K+-channel α-subunit KCNQ1 is expressed in a wide variety of tissues including heart, skeletal muscle, liver, and epithelia. Most recent evidence revealed an association of the KCNQ1 gene with the susceptibility to type 2 diabetes. KCNQ1 participates in the regulation of cell volume, which is, in turn, critically important for the regulation of metabolism by insulin. The present study explored the influence of KCNQ1 on insulin-induced cellular K+ uptake and glucose metabolism. Insulin (100 nM)-induced K+ uptake was determined in isolated perfused livers from KCNQ1-deficient mice ( kcnq1−/−) and their wild-type littermates ( kcnq1+/+). Moreover, plasma glucose and insulin levels, intraperitoneal glucose (3 g/kg) tolerance, insulin (0.15 U/kg)-induced hypoglycemia, and peripheral uptake of radiolabeled 3H-deoxy-glucose were determined in both genotypes. Insulin-stimulated hepatocellular K+ uptake was significantly more sustained in isolated perfused livers from kcnq1−/− mice than from kcnq1+/+mice. The decline of plasma glucose concentration following an intraperitoneal injection of insulin was again significantly more sustained in kcnq1−/− than in kcnq1+/+ mice. Both fasted and nonfasted plasma glucose and insulin concentrations were significantly lower in kcnq1−/− than in kcnq1+/+mice. Following an intraperitoneal glucose injection, the peak plasma glucose concentration was significantly lower in kcnq1−/− than in kcnq1+/+mice. Uptake of 3H-deoxy-glucose into skeletal muscle, liver, kidney and lung tissue was significantly higher in kcnq1−/− than in kcnq1+/+mice. In conclusion, KCNQ1 counteracts the stimulation of cellular K+ uptake by insulin and thereby influences K+-dependent insulin signaling on glucose metabolism. The observations indicate that KCNQ1 is a novel molecule affecting insulin sensitivity of glucose metabolism.

Background: The serum and glucocorticoid inducible kinase SGK1, which was originally cloned from mammary tumor cells, is highly expressed in some but not all tumors. SGK1 confers survival to several tumor cells. Along those lines, the number of colonic tumors following chemical carcinogenesis was decreased in SGK1 knockout mice. Recently, a highly selective SGK inhibitor (EMD638683) has been developed. The present study explored whether EMD638683 affects survival of colon carcinoma cells in vitro and impacts on development of colonic tumors in vivo. Methods: Colon carcinoma (Caco-2) cells were exposed to EMD638683 with or without exposure to radiation (3 Gray) and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V binding, mitochondrial potential from JC-9 fluorescence, caspase 3 activity from CaspGlow Fluorescein staining, DNA degradation from propidium iodide staining as well as late apoptosis from annexin-V FITC and propidium iodide double staining. In vivo tumor growth was determined in wild type mice subjected to chemical carcinogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium in drinking water for 7 days). Results: EMD638683 treatment significantly augmented the radiation-induced decrease of forward scatter, increase of phosphatidylserine exposure, decrease of mitochondrial potential, increase of caspase 3 activity, increase of DNA fragmentation and increase of late apoptosis. The in vivo development of tumors following chemical carcinogenesis was significantly blunted by treatment with EMD638683. Conclusions: EMD638683 promotes radiation-induced suicidal death of colon tumor cells in vitro and decreases the number of colonic tumors following chemical carcinogenesis in vivo.

Background: The economic burden associated with major depressive disorder and anxiety disorders render both disorders the most common and debilitating psychiatric illnesses. To date, the exact cellular and molecular mechanisms underlying the pathophysiology, successful treatment and prevention of these highly associated disorders have not been identified. Akt2 is a key protein in the phosphatidylinositide-3 (PI3K) / glycogen synthase 3 kinase (GSK3) signaling pathway, which in turn is involved in brain-derived neurotrophic factor (BDNF) effects on fear memory, mood stabilisation and action of several antidepressant drugs. The present study thus explored the impact of Akt2 on behaviour of mice. Methods: Behavioural studies (Open-Field, Light-Dark box, O-Maze, Forced Swimming Test, Emergence Test, Object Exploration Test, Morris Water Maze, Radial Maze) have been performed with Akt2 knockout mice (akt^-/-) and corresponding wild type mice (akt^+/+). Results: Anxiety and depressive behavior was significantly higher in akt^-/- than in akt^+/+ mice. The akt^-/- mice were cognitively unimpaired but displayed increased anxiety in several behavioral tests (O-Maze test, Light-Dark box, Open Field test). Moreover, akt^-/- mice spent more time floating in the Forced Swimming test, which is a classical feature of experimental depression. Conclusion: Akt2 might be a key factor in the pathophysiology of depression and anxiety.

SummaryThe serum and glucocorticoid inducible kinase SGK1 was originally cloned from mammary tumor cells. SGK1 was found to be upregulated in a variety of tumors, but down-regulated in several distinct tumors. Thus, evidence for a role of SGK1 in tumor growth remained conflicting. According to in vitro observations, SGK1 is upregulated by the oncogene b-catenin and negatively regulates the proapoptotic transcription factor FOXO3a, which in turn stimulates transcription of the Bcl2-interacting mediator BIM. This study aimed to define the role of SGK1 in colon carcinoma in vivo. SGK1 knockout mice (sgk1 2/2 ) and their wild type littermates (sgk1 1/1 ) were subjected to chemical cancerogenesis (intraperitoneal injection of 20 mg/kg 1,2-dimethylhydrazine followed by three cycles of 30 g/L synthetic dextran sulfate sodium for 7 days). Moreover, SGK1 was silenced in HEK293 cells. FOXO3a and BIM protein abundance was determined by Western blotting and immunohistochemistry. Following chemical cancerogenesis, sgk1 2/2 mice developed significantly less colonic tumors than sgk1 1/1 mice. According to Western blotting and immunohistochemistry, SGK1 deficiency enhanced the expression of FOXO3a and BIM both, in vitro and in vivo. SGK1 deficiency counteracts the development of colonic tumors, an effect at least in part due to up-regulation of FOXO3a and BIM.2009 IUBMB IUBMB Life, 61 (7): [768][769][770][771][772][773][774][775][776] 2009

Background/Aims: Gum arabic (GA) is a Ca²⁺-, Mg²⁺- and K⁺-rich dietary fiber used for the treatment of patients with chronic kidney disease in Middle Eastern countries. In healthy mice, GA treatment increases creatinine clearance, renal ADH excretion, as well as intestinal and renal excretion of Mg²⁺ and Ca²⁺. GA decreases plasma Pi concentration, urinary Pi and Na⁺ excretion. The present study explored the effects of GA on renal function in diabetic mice. Methods: Metabolic cage experiments were performed on Akita mice (akita^+/–), which spontaneously develop insulin deficiency and thus hyperglycemia. Plasma and urinary concentrations of Na⁺, K⁺ and Ca²⁺ were measured by flame photometry (AFM 5051, Eppendorf, Germany), creatinine by the Jaffé method, phosphate photometrically, urea by an enzymatic method, glucose utilizing a glucometer and an enzymatic kit, aldosterone using an RIA, urinary albumin fluorometrically, and blood pressure by the tail-cuff method. Results: GA (10% in drinking water) significantly increased urinary excretion of Ca²⁺ and significantly decreased plasma phosphate and urea concentrations, urinary flow rate, urinary Na⁺, phosphate and glucose excretion, blood pressure and proteinuria. Conclusions: GA treatment decreases blood pressure and proteinuria in diabetic mice and may thus prove beneficial in diabetic nephropathy.