Sarah Steane

Impaired memory and olfactory performance in NaSi-1 sulphate transporter deficient mice

In the present study, NaSi-1 sulphate transporter knock-out (Nas1-/-) mice, an animal model of hyposulphataemia, were examined for spatial memory and learning in a Morris water maze, and for olfactory function in a cookie test. The Nas1-/- mice displayed significantly (P<0.05) increased latencies to find an escape platform in the reversal learning trials at 2 days but not 1 day after the last acquisition trial in a Morris water maze test, suggesting that Nas1-/- mice may have proactive memory interference. While the wild-type (Nas1+/+) mice showed a significant (P<0.02) decrease in time to locate a hidden food reward over four trials after overnight fasting, Nas1-/- mice did not change their performance, resulting in significantly (P<0.05) higher latencies when compared to their Nas1+/+ littermates. There were no significant differences between Nas1-/- and Nas1+/+ mice in the cookie test after moderate food deprivation. In addition, both Nas1-/- and Nas1+/+ mice displayed similar escape latencies in the acquisition phase of the Morris water maze test, suggesting that learning, motivation, vision and motor skills required for the task may not be affected in Nas1-/- mice. This is the first study to demonstrate an impairment in memory and olfactory performance in the hyposulphataemic Nas1-/- mouse.

Behavioural abnormalities of the hyposulphataemic Nas1 knock-out mouse.

We recently generated a sodium sulphate cotransporter knock-out mouse (Nas1-/-) which has increased urinary sulphate excretion and hyposulphataemia. To examine the consequences of disturbed sulphate homeostasis in the modulation of mouse behavioural characteristics, Nas1-/- mice were compared with Nas1+/- and Nas1+/+ littermates in a series of behavioural tests. The Nas1-/- mice displayed significantly (P < 0.001) decreased marble burying behaviour (4.33 +/- 0.82 buried) when compared to Nas1+/+ (7.86 +/- 0.44) and Nas1+/- (8.40 +/- 0.37) animals, suggesting that Nas1-/- mice may have decreased object-induced anxiety. The Nas1-/- mice also displayed decreased locomotor activity by moving less distance (1.53 +/- 0.27 m, P < 0.05) in an open-field test when compared to Nas1+/+ (2.31 +/- 0.24 m) and Nas1+/- (2.15 +/- 0.19 m) mice. The three genotypes displayed similar spatiotemporal and ethological behaviours in the elevated-plus maze and open-field test, with the exception of a decreased defecation frequency by the Nas1-/- mice (40% reduction, P < 0.01). There were no significant differences between Nas1-/- and Nas1+/+ mice in a rotarod performance test of motor coordination and in the forced swim test assessing (anti-)depressant-like behaviours. This is the first study to demonstrate behavioural abnormalities in the hyposulphataemic Nas1-/- mice.

Hyperserotonaemia and reduced brain serotonin levels in NaS1 sulphate transporter null mice.

Sulphate (SO42−) is conjugated to numerous endogenous compounds, including serotonin (5-HT). The NaS1 sulphate transporter is primarily expressed in the kidney, where it maintains blood SO42− concentrations. Previously, we generated NaS1 null (Nas1−/−) mice, which have hyposulphataemia and decreased anxiety and locomotor activity. In this study, we investigated 5-HT and 5-hydroxyindole acetic acid (5-HIAA) concentrations, and 5-HT receptor mRNA levels. Nas1−/− mice exhibited a doubling in blood 5-HT concentration, but a 12% reduction in brain levels of 5-HT and 5-hydroxyindole acetic acid. Brain 5-HT1A and 5-HT1B receptor mRNA levels were increased by 50%, compared with wild-type mice. Our data indicate that decreased circulating SO42− concentrations modulate 5-HT neurotransmitter and receptor levels, in a manner consistent with the behavioural phenotypes of Nas1−/− mice.

Differential mRNA Expression and Glucocorticoid-Mediated Regulation of TRPM6 and TRPM7 in the Heart and Kidney throughout Murine Pregnancy and Development

The transient receptor potential (TRP) channels TRPM6 and TRPM7 are critically involved in maintaining whole body and cellular Mg2+ homeostasis and ensuring the normal function of organs such as the heart and kidney. However, we do not know how the expression of TRPM6 and TPRM7 in these organs changes throughout fetal development and adult life, and whether this expression can be hormonally regulated. This study determined the ontogeny of TRPM6 and TRPM7 mRNA expression from mid-gestation through to adulthood in the mouse. In a second series of experiments, we examined how maternal administration of the glucocorticoids corticosterone and dexamethasone between embryonic days 12.5–15 affected TRPM6 and TRPM7 channel mRNA expression in the mother and fetus. Whilst renal TRPM7 expression was relatively constant throughout development, renal TRPM6 expression was markedly upregulated after birth. In contrast, cardiac TRPM7 expression was 2–4 fold higher in the fetus than in the adult. Surprisingly, TRPM6 expression was detected in the fetal heart (qPCR and in situ hybridization). Glucocorticoid administration during gestation increased fetal cardiac expression of both channels without affecting renal expression. In contrast, in the dam renal TRPM6 and TRPM7 expression was increased by glucocorticoids with no change in the cardiac channel expression. These data suggest that TRPM6 and TRPM7 channels are important in organogenesis, and that elevated maternal glucocorticoid levels can alter the expression of these channels. This suggests that perturbations in hormonal regulatory systems during pregnancy may adversely impact upon normal fetal development, at least in part by altering expression of TRPM channels.

The effects of gestational age and maternal hypoxia on the placental renin angiotensin system in the mouse

INTRODUCTION The renin angiotensin system (RAS) is an important mediator of placental development. However, a comprehensive expression profile for 8 key components of the placental RAS throughout murine gestation has not been performed. Furthermore, maternal hypoxia induces dysregulation of RAS expression in fetal tissues but the effects on the murine placental RAS are less well known. METHODS Placentas were collected from male and female CD1 mouse fetuses at seven gestational ages for qPCR analysis of Agt, Ren1, Atp6ap2, Ace, Ace2, Agtr1a, Agtr2 and Mas1. mRNA localisation of Agtr1 and Mas1 and protein localisation of ACE and ACE2 was determined at E18.5. To determine the effects of maternal hypoxia on the placental RAS, mice were housed in 12% oxygen from E14.5-E18.5 and placentas examined at E18.5. RESULTS All RAS genes were expressed in the placenta throughout pregnancy and expression varied with fetal sex and age. Agtr1 was expressed within the labyrinth while Mas1 was expressed within the intraplacental yolk sac. ACE and ACE2 were localised to both labyrinth and junctional zones. In response to maternal hypoxia the expression of Agt, Ace and Ace2 was decreased but expression of Agtr1a was increased. Ace and Agtr1a mRNA levels were affected to a greater extent in females compared to males. DISCUSSION Collectively, the location within the placenta as well as the expression profiles identified, support a role for the placental RAS in labyrinth development. The placental RAS is disturbed by maternal hypoxia in a sexually dimorphic manner and may contribute to impairment of placental vascular development.

Placental O-GlcNAc-transferase expression and interactions with the glucocorticoid receptor are sex specific and regulated by maternal corticosterone exposure in mice

Maternal stress programs offspring disease in a sexually dimorphic manner with males often more adversely affected. Previous studies of maternal glucocorticoid exposure suggest male vulnerability may derive from placental alterations. The hexosamine signalling pathway and O-linked glycosylation (O-GlcNAcylation) are part of an essential adaptive survival response in healthy cells. The key enzyme involved is O-linked-N-acetylglucosamine transferase (OGT), a gene recently identified as a sex-specific placental biomarker of maternal stress. Using a mouse model of maternal corticosterone (Cort) exposure, we examined components of hexosamine biosynthesis/signalling and O-GlcNAcylation in whole placentae at E14.5. Our results demonstrate sex-specific differences in OGT levels and O-GlcNAcylation during Cort exposure which impacts on key mediators of cell survival, in particular AKT as well as the stress responsive OGT/GR transrepression complex. In male placentae only, Cort exposure increased Akt O-GlcNacylation which correlated with decreased phosphorylation. Female placentae had higher basal OGT and OGT/GR complex compared with male placentae. Cort exposure did not alter these levels in female placentae but increased global O-GlcNacylation. In male placentae Cort increased OGT and OGT/GR complex with no change in global O-GlcNacylation. These findings suggest that sex-specific differences in placental OGT play a key role in the sexually dimorphic responses to stress.

The effects of periconceptional maternal alcohol intake and a postnatal high-fat diet on obesity and liver disease in male and female rat offspring.

The effects of maternal alcohol consumption around the time of conception on offspring are largely unknown and difficult to determine in a human population. This study utilized a rodent model to examine if periconceptional alcohol (PC:EtOH) consumption, alone or in combination with a postnatal high-fat diet (HFD), resulted in obesity and liver dysfunction. Sprague-Dawley rats were fed a control or an ethanol-containing [12.5% (vol/vol) EtOH] liquid diet from 4 days before mating until 4 days of gestation ( n = 12/group). A subset of offspring was fed a HFD between 3 and 8 mo of age. In males, PC:EtOH and HFD increased total body fat mass ( PPC:EtOH < 0.05, PHFD < 0.0001); in females, only HFD increased fat mass ( PHFD < 0.0001). PC:EtOH increased microvesicular liver steatosis in male, but not female, offspring. Plasma triglycerides, HDL, and cholesterol were increased in PC:EtOH-exposed males ( PPC:EtOH < 0.05), and LDL, cholesterol, and leptin (Lep) were increased in PC:EtOH-exposed females ( PPC:EtOH < 0.05). mRNA levels of Tnf-α and Lep in visceral adipose tissue were increased by PC:EtOH in both sexes ( PPC:EtOH < 0.05), and Il-6 mRNA was increased in males ( PPC:EtOH < 0.05). These findings were associated with reduced expression of microRNA-26a, a known regulator of IL-6 and TNF-α. Alcohol exposure around conception increases obesity risk, alters plasma lipid and leptin profiles, and induces liver steatosis in a sex-specific manner. These programmed phenotypes were similar to those caused by a postnatal HFD, particularly in male offspring. These results have implications for the health of offspring whose mothers consumed alcohol around the time of conception.

446 THE TRPM7 CHANNEL-KINASE CONTRIBUTES TO ANGIOTENSIN II-INDUCED HYPERTROPHIC REMODELLING IN CARDIOMYOCYTES

Background and Aims: The transient receptor potential melastatin 7 (TRPM7) channel is a unique protein that contains both a Mg2+ and Ca2+-permeable pore and an active kinase domain. Activity of TRPM7 is essential for cell viability and embryonic development, and inhibition of TRPM7 impairs the growth of vascular cells. In this study we used RNA interference to determine whether TRPM7 could mediate hypertrophic signalling and remodelling in cardiomyocytes. Methods: Primary ventricular cardiomyocytes were isolated from neonatal rats. Multiple short hairpin (shRNAs) were used to downregulate TRPM7 in these cells prior to treatment with angiotensin II (AngII, 0.1 &mgr;M, 48 hours). Hypertrophic signalling was assessed by promoter –driven luciferase reporter assays measuring the activation of the hypertrophy-associated genes myosin light chain 2 v (MLC-2 v), atrial natriuretic peptide (ANP) and cyclin D. Phalloidin staining was used to assess cytoskeletal reorganization of the actin cytoskeleton. Results: AngII caused robust activation of the hypertrophic gene MLC-2 v (465 ± 85 % increase versus untreated control) that was significantly reduced by silencing of TRPM7 (to 135 ± 66 %, 226 ± 43% and 215 ± 78 % of control for three independent shRNAs targeting TRPM7, n = 8, P < 0.05). Similar results were seen for ANP and cyclin D. Silencing of TRPM7 also prevented AngII-induced sacromeric re-organization and cellular hypertrophy. Conclusions: The Mg2+ channel TRPM7 can mediate AngII-induced hypertrophic signalling and remodelling in cardiomyocytes, possibly through the activity of its kinase domain.