Slc20a1 and Slc20a2 regulate neuronal plasticity and cognition independently of their phosphate transport ability.

In recent years, primary familial brain calcification (PFBC), a rare neurological disease characterized by a wide spectrum of cognitive disorders, has been associated to mutations in the sodium (Na)-Phosphate (Pi) co-transporter SLC20A2. However, the functional roles of the Na-Pi co-transporters in the brain remain still largely elusive. Here we show that Slc20a1 (PiT-1) and Slc20a2 (PiT-2) are the most abundant Na-Pi co-transporters expressed in the brain and are involved in the control of hippocampal-dependent learning and memory. We reveal that Slc20a1 and Slc20a2 are differentially distributed in the hippocampus and associated with independent gene clusters, suggesting that they influence cognition by different mechanisms. Accordingly, using a combination of molecular, electrophysiological and behavioral analyses, we show that while PiT-2 favors hippocampal neuronal branching and survival, PiT-1 promotes synaptic plasticity. The latter relies on a likely Otoferlin-dependent regulation of synaptic vesicle trafficking, which impacts the GABAergic system. These results provide the first demonstration that Na-Pi co-transporters play key albeit distinct roles in the hippocampus pertaining to the control of neuronal plasticity and cognition. These findings could provide the foundation for the development of novel effective therapies for PFBC and cognitive disorders.

Acyl-CoA binding protein for the experimental treatment of anorexia.

Extracellular acyl-coenzyme A binding protein [ACBP encoded by diazepam binding inhibitor (DBI)] is a phylogenetically ancient appetite stimulator that is secreted in a nonconventional, autophagy-dependent fashion. Here, we show that low ACBP/DBI plasma concentrations are associated with poor prognosis in patients with anorexia nervosa, a frequent and often intractable eating disorder. In mice, anorexia induced by chronic restraint stress (CRS) is accompanied by a reduction in circulating ACBP/DBI concentrations. We engineered a chemical-genetic system for the secretion of ACBP/DBI through a biotin-activatable, autophagy-independent pathway. In transgenic mice expressing this system in hepatocytes, biotin-induced elevations in plasma ACBP/DBI concentrations prevented anorexia induced by CRS or chemotherapeutic agents including cisplatin, doxorubicin, and paclitaxel. ACBP/DBI reversed the CRS or cisplatin-induced increase in plasma lipocalin-2 concentrations and the hypothalamic activation of anorexigenic melanocortin 4 receptors, for which lipocalin-2 is an agonist. Daily intravenous injections of recombinant ACBP/DBI protein or subcutaneous implantation of osmotic pumps releasing recombinant ACBP/DBI mimicked the orexigenic effects of the chemical-genetic system. In conclusion, the supplementation of extracellular and peripheral ACBP/DBI might constitute a viable strategy for treating anorexia.

Antidepressants reverse the attenuation of the neurotrophic MEK/MAPK cascade in frontal cortex by elevated platform stress; reversal of effects on LTP is associated with GluA1 phosphorylation.

Exposure to stress causes dysfunctions in circuits connecting hippocampus and prefrontal cortex (H-PFC). Long term potentiation (LTP) induced in vivo in rats at H-PFC synapses is impaired by acute elevated platform stress in a manner that can be restored by treatment with certain antidepressants. To identify biochemical pathways in rat frontal cortex underlying this stress-mediated impairment of synaptic plasticity, we examined the phosphorylation state of receptors, signaling proteins and transcription factors implicated in neuronal plasticity. Transient changes in the phosphorylation states of Ser217/221-MEK, Thr183/Tyr185-p42MAPK, Thr202/Tyr204-p44MAPK, Thr180/Tyr182-p38MAPK, Thr218/Tyr220-ERK5, Thr308-Akt, Ser63-ATF-1, Ser1303-GluN2B, Tyr490/515-TrkA/B were found. BDNF was down-regulated after elevated platform stress suggesting that it could regulate the MEK/MAPK signaling cascade. Acute treatment with the antidepressants tianeptine and imipramine reversed the stress-induced down-regulation of P-Ser217/221-MEK. However, stress-induced impairment of H-PFC LTP was only restored by acute treatment with tianeptine and not by imipramine. Tianeptine, but not imipramine, increased the phosphorylation of Ser831-GluA1. Altogether, these results indicate that acute elevated platform stress down-regulates a putative BDNF/MEK/MAPK signaling cascade in the frontal cortex in a manner that is reversible by the antidepressants tianeptine and imipramine. Moreover, changes in LTP may be associated with phosphorylation of AMPA receptors and with some specificity for certain antidepressants. Indeed, stress-induced impairment of H-PFC LTP was only restored by acute treatment with tianeptine and not by imipramine. Tianeptine, but not imipramine, increased the phosphorylation of Ser831-GluA1, indicating a potential effect on AMPA receptor phosphorylation being involved in the reversal of LTP.

The prefrontal cortex as a key target of the maladaptive response to stress.

Research on the detrimental effects of stress in the brain has mainly focused on the hippocampus. Because prefrontal cortex (PFC) dysfunction characterizes many stress-related disorders, we here analyzed the impact of chronic stress in rats on the integrity of the hippocampal-PFC pathway, monitored by behavioral and electrophysiological function and morphological assessment. We show that chronic stress impairs synaptic plasticity by reducing LTP induction in the hippocampal-PFC connection; in addition, it induces selective atrophy within the PFC and severely disrupts working memory and behavioral flexibility, two functions that depend on PFC integrity. We also demonstrate that short periods of stress exposure induce spatial reference memory deficits before affecting PFC-dependent tasks, thus suggesting that the impairment of synaptic plasticity within the hippocampus-to-PFC connection is of relevance to the stress-induced PFC dysfunction. These findings evidence a fundamental role of the PFC in maladaptive responses to stress and identify this area as a target for intervention in stress-related disorders.

Cellular knock-down of quinone reductase 2: a laborious road to successful inhibition by RNA interference.

NRH:quinone oxidoreductase 2 (QR2) is a long forgotten oxidoreductive enzyme that metabolizes quinones and binds melatonin. We used the potency of the RNA interference (RNAi)-mediated gene silencing to build a cellular model in which the role of QR2 could be studied. Because standard approaches were poorly successful, we successively used: (1) two chemically synthesized fluorescent small interfering RNA (siRNA) duplexes designed and tested for their gene silencing capacity leading to a maximal 40% QR2 gene silencing 48h post-transfection; (2) double transfection and cell-sorting of high fluorescent siRNA-transfected HT22 cells further enhancing QR2 RNAi silencing to 88%; (3) stable QR2 knock-down HT22 cell lines established with H1and U6 promoter driven QR2 short hairpin RNA (shRNA) encoding vectors, resulting in a 71-80% reduction of QR2 enzymatic activity in both QR2 shRNA HT22 cells. Finally, as a first step in the study of this cellular model, we observed a 42-48% reduction of menadione/BNAH-mediated toxicity in QR2 shRNA cells compared to the wild-type HT22 cells. Although becoming widespread and in some cases effective, siRNA-mediated cellular knock-down proves in the present work to be of marginal efficiency. Much development is required for this technique to be of general application.

Common efficacy of psychotropic drugs in restoring stress-induced impairment of prefrontal plasticity.

We recently investigated the effects of stress on synaptic plasticity in the prefrontal cortex, namely the prelimbic area or the apparent homologue of the primate subgenual prefrontal cortex in humans where most of the hippocampal terminal fields are localized. Exposure to an acute stress causes a remarkable and long-lasting inhibition of long term potentiation (LTP) in the frontal cortex evoked by stimulation of hippocampal outflow and this impairment is prevented by the glucocorticoid receptor antagonist mifepristone. Thus, the frontal cortex is also a target for glucocorticoids involved in the stress response. Current data show that antidepressants of various types, i.e., tianeptine and fluoxetine, at doses normally used in antidepressant testing, restore LTP impaired by prior acute stress. Interestingly, clozapine administered in a similar way after stress rapidly reverses the stress-induced impairment of LTP at doses which do not affect LTP alone. This stress paradigm highlights comorbidity for both etiology and treatment of psychiatric disorders like depression and schizophrenia. Restoring appropriate cognitive functions in circuits associated with dysfunctions in coping with stress may be proposed as a new systems-level approach to drug discovery and development. We are presently investigating the involvement of signalling molecules in producing these plastic changes.

Characterization of the melatoninergic MT3 binding site on the NRH:quinone oxidoreductase 2 enzyme.

Melatonin acts through a series of molecular targets: the G-protein coupled receptors, MT1 and MT2, and a third binding site, MT3, recently identified as the enzyme NRH:quinone oxydoreductase 2 (QR2). The relationship between the multiple physiological functions of melatonin and this enzyme remains unclear. Because of the relationship of QR2 with the redox status of cells, these studies could bring the first tools for a molecular rationale of the antioxidant effects of melatonin. In the present paper, we used a QR2-stably expressing cell line and hamster kidneys to compare the 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine binding data, and to characterize the MT3 binding site. We designed and tested compounds from two distinct chemicals series in a displacement assay of the two MT3 ligands, 2-[125I]-iodomelatonin and 2-[125I]-iodo-5-methoxycarbonylamino-N-acetyltryptamine from their cloned target. We also tested their ability to inhibit QR2 catalytic activity. These compounds were separated into two classes: those that bind within the catalytic site (and being inhibitors) and those that bind outside it (and therefore not being inhibitors). Compounds range from potent ligands (K(i) = 1 nM) to potent inhibitors (14 nM), and include one compound [NMDPEF: N-[2-(2-methoxy-6H-dipyrido[2,3-a:3,2-e]pyrrolizin-11-yl)ethyl]-2-furamide] active on both parameters in the low nanomolar range. To dissect the physio-pathological pathways in which QR2, MT3 and melatonin meet, one needs more compounds binding to MT3 and/or inhibitors of QR2 enzymatic activity. The compounds described in the present paper are new tools for such a task.

Organs from mice deleted for NRH:quinone oxidoreductase 2 are deprived of the melatonin binding site MT3.

Two melatonin receptors (MT1 and MT2) have been cloned. A third melatonin binding site, MT3, is known with remarkable and distinct pharmacological properties. We previously reported the purification of MT3 and identified it as the enzyme dihydronicotinamide riboside:quinone reductase 2 (NQO2). To investigate the relationship between NQO2 and MT3, we generated a NQO2-/- mouse strain. These mice no longer present MT3 binding sites as measured with 2-[125I]-iodo, 5-methoxycarbonylamino-N-acetyltryptamine, the specific MT3 radioligand. These data establish NQO2 as part of the MT3 binding sites in vivo and resolve the matter of the nature of the third melatonin binding site.

Molecular pharmacology of the ovine melatonin receptor: comparison with recombinant human MT1 and MT2 receptors.

The variations of the pharmacological properties of melatonin receptors between different mammalian species in transfected cell lines have been poorly investigated. In the present study, melatonin analogues have been used to characterize the pharmacology of the recombinant ovine melatonin receptor (oMT1) expressed in CHO cell lines and the native oMT1 from the pars tuberalis (PT). Studies with selective ligands on native and transfected oMT1 showed similar properties for binding affinities [r2(PT/CHO) = 0.85]. The affinities and the functional activities of these ligands were compared with the human receptors (hMT1 or hMT2) expressed in CHO cells as well. The oMT1 and hMT1 receptors had similar pharmacological profiles (r2=0.82). Nevertheless, some of the selective compounds at the human receptor presented a reduced affinity at the ovine receptor. Furthermore, some compounds showed marked different functional activities at oMT1 vs. hMT1 receptors. Our findings demonstrated differences in the pharmacological properties of melatonin receptors in ovine and human species.

New selective ligands of human cloned melatonin MT1 and MT2 receptors.

Melatonin has a key role in the circadian rhythm relay to periphery organs. Melatonin exerts its multiple roles mainly through two seven transmembrane domain, G-coupled receptors, namely MT1 or MT2 receptors. A pharmacological characterization of these human cloned melatonin hMT1 and hMT2 receptors stably expressed in HEK-293 or CHO cells is presented using a 2-[125I]-iodo-melatonin binding assay and a [35S]-GTPgammaS functional assay. Both reference compounds and new chemically diverse ligands were evaluated. Binding affinities at each receptor were found to be comparable on either HEK-293 or CHO cell membranes. Novel non-selective or selective hMT1 and hMT2 ligands are described. The [35S]-GTPgammaS functional assay was used to define the functional activity of these compounds which included partial, full agonist and/or antagonist activity. None of the compounds acted as an inverse agonist. We report new types of selective antagonists, such as S 25567 and S 26131 for MT1 and S 24601 for MT2. These studies brought other new molecular tools such as the selective MT1 agonist, S 24268, as well as the non-selective antagonist, S 22153. Finally, we also discovered S 25150, the most potent melatonin receptor agonist, so far reported in the literature.

Acute stress induces contrasting changes in AMPA receptor subunit phosphorylation within the prefrontal cortex, amygdala and hippocampus.

Exposure to stress causes differential neural modifications in various limbic regions, namely the prefrontal cortex, hippocampus and amygdala. We investigated whether α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) phosphorylation is involved with these stress effects. Using an acute inescapable stress protocol with rats, we found opposite effects on AMPA receptor phosphorylation in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DH) compared to the amygdala and ventral hippocampus (VH). After stress, the phosphorylation of Ser831-GluA1 was markedly decreased in the mPFC and DH, whereas the phosphorylation of Ser845-GluA1 was increased in the amygdala and VH. Stress also modulated the GluA2 subunit with a decrease in the phosphorylation of both Tyr876-GluA2 and Ser880-GluA2 residues in the amygdala, and an increase in the phosphorylation of Ser880-GluA2 in the mPFC. These results demonstrate that exposure to acute stress causes subunit-specific and region-specific changes in glutamatergic transmission, which likely lead to the reduced synaptic efficacy in the mPFC and DH and augmented activity in the amygdala and VH. In addition, these findings suggest that modifications of glutamate receptor phosphorylation could mediate the disruptive effects of stress on cognition. They also provide a means to reconcile the contrasting effects that stress has on synaptic plasticity in these regions. Taken together, the results provide support for a brain region-oriented approach to therapeutics.

Protection of stress-induced impairment of hippocampal/prefrontal LTP through blockade of glucocorticoid receptors: implication of MEK signaling.

We previously reported that exposure to acute and chronic stress impairs long-term potentiation (LTP) in the hippocampal-prefrontal cortex pathway and showed evidence for a fundamental role of the prefrontal cortex in maladaptive responses to stress. The goal of the current studies was to examine whether blockade of glucocorticosteroid receptors (GR), by mifepristone (a Type II glucocorticoid receptor antagonist), just after exposure to acute stress could prevent stress-induced impairment of prefrontal LTP. We further examine the effects of mifepristone on mitogen-activated protein/ERK kinase (MEK) signaling pathway in the prefrontal cortex. The data show that an acute injection of mifepristone after stress restored the stress-induced blockade of prefrontal LTP and reduction of phospho-Ser217/221-MEK. These findings have significance for the treatment of memory deficits in hypercortisolemic states, such as stress and depression.