Rosamaria Orlando

Nanomolar concentrations of anabolic-androgenic steroids amplify excitotoxic neuronal death in mixed mouse cortical cultures.

The use of anabolic-androgenic steroids (AASs) in the world of sport has raised a major concern for the serious, sometimes life-threatening, side effects associated with these drugs. Most of the CNS effects are of psychiatric origin, and whether or not AASs are toxic to neurons is yet unknown. We compared the effect of testosterone with that of the AASs, 19-nortestosterone (nandrolone), stanozolol, and gestrinone, on excitotoxic neuronal death induced by N-methyl-d-aspartate (NMDA) in primary cultures of mouse cortical cells. In the most relevant experiments, steroids were applied to the cultures once daily during the 4 days preceding the NMDA pulse. Under these conditions, testosterone amplified excitotoxic neuronal death only at very high concentrations (10 muM), whereas it was protective at concentrations of 10 nM and inactive at intermediate concentrations. Low concentrations of testosterone became neurotoxic in the presence of the aromatase inhibitors, i.e. anastrozole and aminoglutethimide, suggesting that the intrinsic toxicity of testosterone was counterbalanced by its aromatization into 17beta-estradiol. As opposed to testosterone, nortestosterone, stanozolol and gestrinone amplified NMDA toxicity at nanomolar concentrations; their action was insensitive to aromatase inhibitors, but was abrogated by the androgen receptor antagonist, flutamide. None of the AASs were toxic in the absence of NMDA. These data suggest that AASs increase neuronal vulnerability to an excitotoxic insult and may therefore facilitate neuronal death associated with acute or chronic CNS disorders.

Induction of the Wnt antagonist Dickkopf-1 is involved in stress-induced hippocampal damage.

The identification of mechanisms that mediate stress-induced hippocampal damage may shed new light into the pathophysiology of depressive disorders and provide new targets for therapeutic intervention. We focused on the secreted glycoprotein Dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway, involved in neurodegeneration. Mice exposed to mild restraint stress showed increased hippocampal levels of Dkk-1 and reduced expression of β-catenin, an intracellular protein positively regulated by the canonical Wnt signalling pathway. In adrenalectomized mice, Dkk-1 was induced by corticosterone injection, but not by exposure to stress. Corticosterone also induced Dkk-1 in mouse organotypic hippocampal cultures and primary cultures of hippocampal neurons and, at least in the latter model, the action of corticosterone was reversed by the type-2 glucocorticoid receptor antagonist mifepristone. To examine whether induction of Dkk-1 was causally related to stress-induced hippocampal damage, we used doubleridge mice, which are characterized by a defective induction of Dkk-1. As compared to control mice, doubleridge mice showed a paradoxical increase in basal hippocampal Dkk-1 levels, but no Dkk-1 induction in response to stress. In contrast, stress reduced Dkk-1 levels in doubleridge mice. In control mice, chronic stress induced a reduction in hippocampal volume associated with neuronal loss and dendritic atrophy in the CA1 region, and a reduced neurogenesis in the dentate gyrus. Doubleridge mice were resistant to the detrimental effect of chronic stress and, instead, responded to stress with increases in dendritic arborisation and neurogenesis. Thus, the outcome of chronic stress was tightly related to changes in Dkk-1 expression in the hippocampus. These data indicate that induction of Dkk-1 is causally related to stress-induced hippocampal damage and provide the first evidence that Dkk-1 expression is regulated by corticosteroids in the central nervous system. Drugs that rescue the canonical Wnt pathway may attenuate hippocampal damage in major depression and other stress-related disorders.

Molecular pharmacodynamics of new oral drugs used in the treatment of multiple sclerosis

New oral drugs have considerably enriched the therapeutic armamentarium for the treatment of multiple sclerosis. This review focuses on the molecular pharmacodynamics of fingolimod, dimethyl fumarate (BG-12), laquinimod, and teriflunomide. We specifically comment on the action of these drugs at three levels: 1) the regulation of the immune system; 2) the permeability of the blood–brain barrier; and 3) the central nervous system. Fingolimod phosphate (the active metabolite of fingolimod) has a unique mechanism of action and represents the first ligand of G-protein-coupled receptors (sphingosine-1-phosphate receptors) active in the treatment of multiple sclerosis. Dimethyl fumarate activates the nuclear factor (erythroid-derived 2)-related factor 2 pathway of cell defense as a result of an initial depletion of reduced glutathione. We discuss how this mechanism lies on the border between cell protection and toxicity. Laquinimod has multiple (but less defined) mechanisms of action, which make the drug slightly more effective on disability progression than on annualized relapse rate in clinical studies. Teriflunomide acts as a specific inhibitor of the de novo pyrimidine biosynthesis. We also discuss new unexpected mechanisms of these drugs, such as the induction of brain-derived neurotrophic factor by fingolimod and the possibility that laquinimod and teriflunomide regulate the kynurenine pathway of tryptophan metabolism.

Antidepressant activity of fingolimod in mice.

Recent findings indicate that fingolimod, the first oral drug approved for the treatment of multiple sclerosis (MS), acts as a direct inhibitor of histone deacetylases (HDACs) and enhances the production of brain‐derived neurotrophic factor (BDNF) in the CNS. Both mechanisms are relevant to the pathophysiology and treatment of major depression. We examined the antidepressant activity of fingolimod in mice subjected to chronic unpredictable stress (CUS), a model of reactive depression endowed with face and pharmacological validity. Chronic treatment with fingolimod (3 mg kg−1, i.p., once a day for 4 weeks) reduced the immobility time in the forced swim test (FST) in a large proportion of CUS mice. This treatment also caused anxiogenic‐like effects in the social interaction test without affecting anxiety‐like behavior in the elevated plus maze or spatial learning in the water maze. CUS mice showed reduced BDNF levels and enhanced HDAC2 levels in the hippocampus. These changes were reversed by fingolimod exclusively in mice that showed a behavioral response to the drug in the FST. Fingolimod treatment also enhanced H3 histone K14‐acetylation and adult neurogenesis in the hippocampus of CUS mice. Fingolimod did not affect most of the parameters we have tested in unstressed control mice. The antidepressant‐like activity of fingolimod was confirmed in mice chronically treated with corticosterone. These findings show for the first time that fingolimod exerts antidepressant‐like effect acting in a “disease‐dependent” manner, and raise the interesting possibility that the drug could relieve depressive symptoms in MS patients independently of its disease‐modifying effect on MS.

Cigarette smoke inhibits adenine nucleotide hydrolysis by human platelets

Cigarette smoking is a recognized risk factor for cardiovascular diseases and has been implicated in the pathogenesis of atherosclerosis and thrombotic events. In athero-thrombotic diseases, the extracellular adenine nucleotides play an important role by triggering a range of effects such as the recruitment and activation of platelets, endothelial cell activation and vasoconstriction. NTPDase, a plasma membrane-bound enzyme, is the most relevant enzyme involved in the hydrolysis of extracellular tri- and di-phosphate nucleotides to adenosine monophosphate, which is further degraded by 5′ectonucleotidase to the anti-thrombotic and anti-inflammatory mediator adenosine. Thus, the preserved activity of these enzymes, regulating the extracellular concentrations of nucleotides, is critical in thromboregulatory functions. In the present in vitro study, performed on human platelets suspended in undiluted or diluted aqueous cigarette smoke extract (aCSE), we demonstrated that undiluted and 1 : 2 diluted aCSE is able to significantly reduce ADP hydrolysis (−24% and 12%, respectively) by intact human platelets. ATP degradation was also reduced (−31%) by undiluted aCSE. Conversely, aCSE did not alter platelet AMP hydrolysis. Results obtained by using N-acetylcysteine, a thiol-containing antioxidant, suggest that stable oxidants present in aCSE are responsible for the platelet NTPDase inhibition induced by aCSE. The decreased adenine nucleotide degradation could play a significant role in the extensive platelet activation and vascular inflammation observed in chronic smokers.

Exposure to predator odor and resulting anxiety enhances the expression of the α2δ subunit of voltage-sensitive calcium channels in the amygdala

The α2δ subunit of voltage‐sensitive calcium channels (VSCCs) is the molecular target of pregabalin and gabapentin, two drugs marked for the treatment of focal epilepsy, neuropathic pain, and anxiety disorders. Expression of the α2δ subunit is up‐regulated in the dorsal horns of the spinal cord in models of neuropathic pain, suggesting that plastic changes in the α2δ subunit are associated with pathological states. Here, we examined the expression of the α2δ‐1 subunit in the amygdala, hippocampus, and frontal cortex in the trimethyltiazoline (TMT) mouse model of innate anxiety. TMT is a volatile molecule present in the feces of the rodent predator, red fox. Mice that show a high defensive behavior during TMT exposure developed anxiety‐like behavior in the following 72 h, as shown by the light–dark test. Anxiety was associated with an increased expression of the α2δ‐1 subunit of VSCCs in the amygdaloid complex at all times following TMT exposure (4, 24, and 72 h). No changes in the α2δ‐1 protein levels were seen in the hippocampus and frontal cortex of mice exposed to TMT. Pregabalin (30 mg/kg, i.p.) reduced anxiety‐like behavior in TMT‐exposed mice, but not in control mice. These data offer the first demonstration that the α2δ‐1 subunit of VSCCs undergoes plastic changes in a model of innate anxiety, and supports the use of pregabalin as a disease‐dependent drug in the treatment of anxiety disorders.

Functional partnership between mGlu3 and mGlu5 metabotropic glutamate receptors in the central nervous system

ABSTRACT mGlu5 receptors are involved in mechanisms of activity‐dependent synaptic plasticity, and are targeted by drugs developed for the treatment of CNS disorders. We report that mGlu3 receptors, which are traditionally linked to the control of neurotransmitter release, support mGlu5 receptor signaling in neurons and largely contribute to the robust mGlu5 receptor‐mediated polyphosphoinositide hydrolysis in the early postnatal life. In cortical pyramidal neurons, mGlu3 receptor activation potentiated mGlu5 receptor‐mediated somatic Ca2+ mobilization, and mGlu3 receptor‐mediated long‐term depression in the prefrontal cortex required the endogenous activation of mGlu5 receptors. The interaction between mGlu3 and mGlu5 receptors was also relevant to mechanisms of neuronal toxicity, with mGlu3 receptors shaping the influence of mGlu5 receptors on excitotoxic neuronal death. These findings shed new light into the complex role played by mGlu receptors in physiology and pathology, and suggest reconsideration of some of the current dogmas in the mGlu receptor field. HIGHLIGHTSmGlu5 and mGlu3 receptors functionally interact in the central nervous system.mGlu3 receptors support mGlu5 receptor signaling in the early postnatal life.mGlu3 receptor activation potentiate mGlu5 receptor‐mediated somatic Ca2+ mobilization.mGlu3 and mGlu5 receptors interact to induce LTD in the prefrontal cortex.This interaction is relevant to mechanisms of neuronal toxicity.

Stress as risk factor for Alzheimer’s disease

&NA; Prolonged stress predisposes susceptible individuals to a number of physiological disorders including cardiovascular disease, obesity and gastrointestinal disorders, as well as psychiatric and neurodegenerative disorders. Preclinical studies have suggested that manipulation of the glucocorticoid milieu can trigger cellular, molecular and behavioral derangement resembling the hallmarks of Alzheimers Disease (AD). For example, stress or glucocorticoid administration can increase amyloid ß precursor protein and tau phosphorylation which are involved in synaptic dysfunction and neuronal death associated with AD. Although since AD was first described in 1906 at a conference in Tubingen, Germany by Alois Alzheimer our knowledge of neuropathological and neurochemical alterations of AD has been impressively increased, at present, pharmacotherapy is symptomatic at best and has no influence on the progression of the disorder. It is generally believed that most of the drugs developed as disease modifiers have failed in clinical trials because treatment started too late, i.e., after the clinical onset of AD. Because AD pathology begins several years prior to the clinical diagnosis, it is imperative to identify subjects at high risk to develop the disorder. Consequently, the search for putative risk factors has gained importance. ApoE4, diabetes/metabolic syndrome, cardiovascular disorders, and a low cognitive reserve are established risk factors for AD. The focus of this review is on stress and glucocorticoids as potential factors increasing the risk to develop AD. Graphical abstract Figure. No caption available.

Type-7 metabotropic glutamate receptors negatively regulate α1-adrenergic receptor signalling.

&NA; We studied the interaction between mGlu7 and &agr;1‐adrenergic receptors in heterologous expression systems, brain slices, and living animals. L‐2‐Amino‐4‐phosphonobutanoate (L‐AP4), and l‐serine‐O‐phosphate (L‐SOP), which activate group III mGlu receptors, restrained the stimulation of polyphosphoinositide (PI) hydrolysis induced by the &agr;1‐adrenergic receptor agonist, phenylephrine, in HEK 293 cells co‐expressing &agr;1‐adrenergic and mGlu7 receptors. The inibitory action of L‐AP4 was abrogated by (i) the mGlu7 receptor antagonist, XAP044; (ii) the C‐terminal portion of type‐2 G protein coupled receptor kinase; and (iii) the MAP kinase inhibitors, UO126 and PD98059. This suggests that the functional interaction between mGlu7 and &agr;1‐adrenergic receptors was mediated by the &bgr;&ggr;‐subunits of the Gi protein and required the activation of the MAP kinase pathway. Remarkably, activation of neither mGlu2 nor mGlu4 receptors reduced &agr;1‐adrenergic receptor‐mediated PI hydrolysis. In mouse cortical slices, both L‐AP4 and L‐SOP were able to attenuate norepinephrine‐ and phenylephrine‐stimulated PI hydrolysis at concentrations consistent with the activation of mGlu7 receptors. L‐AP4 failed to affect norepinephrine‐stimulated PI hydrolysis in cortical slices from mGlu7−/− mice, but retained its inhibitory activity in slices from mGlu4−/− mice. At behavioural level, i.c.v. injection of phenylephrine produced antidepressant‐like effects in the forced swim test. The action of phenylephrine was attenuated by L‐SOP, which was inactive per se. Finally, both phenylephrine and L‐SOP increased corticosterone levels in mice, but the increase was halved when the two drugs were administered in combination. Our data demonstrate that &agr;1‐adrenergic and mGlu7 receptors functionally interact and suggest that this interaction might be targeted in the treatment of stress‐related disorders. HighlightsmGlu7 receptors and &agr;1‐adrenergic receptors functionally interact.The interaction is shown in heterologous expression systems, brain tissue and living mice.This interaction is mediated by the &bgr;&ggr;‐subunits of the Gi proteins.The interaction requires the activation of the MAP kinase pathway.This receptor cross‐talk might be targeted in the treatment of stress related disorders.

In Vivo Non-radioactive Assessment of mGlu5 Receptor-Activated Polyphosphoinositide Hydrolysis in Response to Systemic Administration of a Positive Allosteric Modulator

mGlu5 receptor-mediated polyphosphoinositide (PI) hydrolysis is classically measured by determining the amount of radioactivity incorporated in inositolmonophosphate (InsP) after labeling of membrane phospholipids with radioactive inositol. Although this method is historically linked to the study of mGlu receptors, it is inappropriate for the assessment of mGlu5 receptor signaling in vivo. Using a new ELISA kit we showed that systemic treatment with the selective positive allosteric modulator (PAM) of mGlu5 receptors VU0360172 enhanced InsP formation in different brain regions of CD1 or C57Black mice. The action of VU0360172 was sensitive to the mGlu5 receptor, negative allosteric modulator (NAM), MTEP, and was abolished in mice lacking mGlu5 receptors. In addition, we could demonstrate that endogenous activation of mGlu5 receptors largely accounted for the basal PI hydrolysis particularly in the prefrontal cortex. This method offers opportunity for investigation of mGlu5 receptor signaling in physiology and pathology, and could be used for the functional screening of mGlu5 receptor PAMs in living animals.