Behavioural and expressional phenotyping of nitric oxide synthase-I knockdown animals.

The gaseous messenger nitric oxide (NO) has been implicated in a wide range of behaviors, including aggression, anxiety, depression, and cognitive functioning. To further elucidate the physiological role of NO and its down-stream mechanisms, we conducted behavioral and expressional phenotyping of mice lacking the neuronal isoform of nitric oxide synthase (NOS-I), the major source of NO in the central nervous system. No differences were observed in activity-related parameters; in contrast to the a priori hypothesis, derived from pharmacological treatments, depression-related tests (Forced Swim Test, Learned Helplessness) also yielded no significantly different results. A subtle anxiolytic phenotype however was present, with knockdown mice displaying a higher open arm time as compared to their respective wildtypes, yet all other investigated anxiety-related parameters were unchanged. The most prominent feature however was gender-independent cognitive impairment in spatial learning and memory, as assessed by the Water Maze test and an automatized holeboard paradigm. No significant dysregulation of monoamine transporters was evidenced by qRT PCR. To further examine the underlying molecular mechanisms, the transcriptome of knockdown animals was thus examined in the hippocampus, striatum and cerebellum by microarray analysis. A set of >120 differentially expressed genes was identified, whereat the hippocampus and the striatum showed similar expressional profiles as compared to the cerebellum in hierarchical clustering. Among the most significantly up-regulated genes were Peroxiredoxon 3, Atonal homologue 1, Kcnj1, Kcnj8, CCAAT/enhancer binding protein (C/EBP), alpha, 3 genes involved in GABA(B) signalling and, intriguingly, the glucocorticoid receptor GR. While GABAergic genes might underlie reduced anxiety, dysregulation of the glucocorticoid receptor can well contribute to a blunted stress response as found in NOS1 knockdown mice. Furthermore, by CREB inhibition, glucocorticoid receptor upregulation could at least partially explain cognitive deficits in these animals. Taken together, NOS1 knockdown mice display a characteristic behavioural profile consisting of reduced anxiety and impaired learning and memory, paralleled by differential expression of the glucocorticoid receptor and GABAergic genes. Further research has to assess the value of these mice as animal models e.g. for Alzheimer's disease or attention deficit disorder, in order to clarify a possible pathophysiological role of NO therein.

Glucocorticoids induce G1 as well as S-phase lengthening in normal human stimulated lymphocytes: differential effects on cell cycle regulatory proteins.

In order to analyze dexamethasone effects on peripheral blood lymphocyte proliferation, we defined various experimental conditions: dexamethasone introduced (i) at the time of phytohemagglutinin stimulation, (ii) 48 h after the beginning of phytohemagglutinin stimulation, and (iii) on unstimulated lymphocytes. In stimulated lymphocytes, we observed an early G1 accumulation (P < 0.005), a delayed increase in the duration of S-phase (P < 0.03), and a consequent increase in cell-cycle duration. The expression of several cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) was modified. Cyclin D3, CDK4, and CDK6 involved in G1-phase control were significantly decreased under dexamethasone treatment whatever the level of stimulation of lymphocytes (stimulated or unstimulated PBL). Cyclin E and CDK2, acting in G1/ S-phase transition and S-phase regulation, decreased in stimulated lymphocytes before any modification of S-phase (P < 0.002). The expression of CKIs, mainly of p27Kip1, appeared to vary with the degree of cell stimulation: a decrease was observed on treated unstimulated lymphocytes, while p27Kip1 increased in dexamethasone-treated cells during stimulation. Our results indicate sequential modifications of the cell-cycle regulation by dexamethasone starting with an action on G1 followed by S-phase control modifications. The protein analysis pinpoints the major complexes concerned: CDK4 and CDK6/cyclin D are mainly involved in G1-phase modifications, while CDK2 and its partner, cyclin E, might be specifically involved in the lengthening of S-phase. The variations observed for p27Kip1 might amplify the functional effects of dexamethasone on kinasic complexes.