Selenoprotein T Exerts an Essential Oxidoreductase Activity That Protects Dopaminergic Neurons in Mouse Models of Parkinson's Disease.

AIMS: Oxidative stress is central to the pathogenesis of Parkinson's disease (PD), but the mechanisms involved in the control of this stress in dopaminergic cells are not fully understood. There is increasing evidence that selenoproteins play a central role in the control of redox homeostasis and cell defense, but the precise contribution of members of this family of proteins during the course of neurodegenerative diseases is still elusive. RESULTS: We demonstrated first that selenoprotein T (SelT) whose gene disruption is lethal during embryogenesis, exerts a potent oxidoreductase activity. In the SH-SY5Y cell model of dopaminergic neurons, both silencing and overexpression of SelT affected oxidative stress and cell survival. Treatment with PD-inducing neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or rotenone triggered SelT expression in the nigrostriatal pathway of wild-type mice, but provoked rapid and severe parkinsonian-like motor defects in conditional brain SelT-deficient mice. This motor impairment was associated with marked oxidative stress and neurodegeneration and decreased tyrosine hydroxylase activity and dopamine levels in the nigrostriatal system. Finally, in PD patients, we report that SelT is tremendously increased in the caudate putamen tissue. INNOVATION: These results reveal the activity of a novel selenoprotein enzyme that protects dopaminergic neurons against oxidative stress and prevents early and severe movement impairment in animal models of PD. CONCLUSIONS: Our findings indicate that selenoproteins such as SelT play a crucial role in the protection of dopaminergic neurons against oxidative stress and cell death, providing insight into the molecular underpinnings of this stress in PD.

Selenoprotein T Deficiency Leads to Neurodevelopmental Abnormalities and Hyperactive Behavior in Mice.

Selenoprotein T (SelT) is a newly discovered thioredoxin-like protein, which is abundantly but transiently expressed in the neural lineage during brain ontogenesis. Because its physiological function in the brain remains unknown, we developed a conditional knockout mouse line (Nes-Cre/SelTfl/fl) in which SelT gene is specifically disrupted in nerve cells. At postnatal day 7 (P7), these mice exhibited reduced volume of different brain structures, including hippocampus, cerebellum, and cerebral cortex. This phenotype, which is observed early during the first postnatal week, culminated at P7 and was associated with increased loss of immature neurons but not glial cells, through apoptotic cell death. This phenomenon was accompanied by elevated levels of intracellular reactive oxygen species, which may explain the increased neuron demise and reduced brain structure volumes. At the second postnatal week, an increase in neurogenesis was observed in the cerebellum of Nes-Cre/SelTfl/fl mice, suggesting the occurrence of developmental compensatory mechanisms in the brain. In fact, the brain volume alterations observed at P7 were attenuated in adult mice. Nevertheless, SelT mutant mice exhibited a hyperactive behavior, suggesting that despite an apparent morphological compensation, SelT deficiency leads to cerebral malfunction in adulthood. Altogether, these results demonstrate that SelT exerts a neuroprotective role which is essential during brain development, and that its loss impairs mice behavior.

Acute and subchronic treatments with selective serotonin reuptake inhibitors increase Nociceptin/Orphanin FQ (NOP) receptor density in the rat dorsal raphe nucleus; interactions between nociceptin/NOP system and serotonin.

Nociceptin/Orphanin FQ is the endogenous ligand of NOP receptor, formerly referred to as the Opioid Receptor-Like 1 receptor. We have previously shown that NOP receptors were located on serotonergic neurons in the rat dorsal raphe nucleus, suggesting possible direct interactions between nociceptin and serotonin in this region, which is a target for antidepressant action. In the present study, we investigated further the link between Selective Serotonin Reuptake Inhibitor (SSRI) antidepressant treatments and the nociceptin/NOP receptor system. Intraperitoneal administration of the SSRI citalopram induced an increase in NOP-receptor density, measured by autoradiographic [(3)H] nociceptin binding, in the rat dorsal raphe nucleus, from the first to the 21st day of treatment. This effect was also observed with other SSRIs (sertraline, fluoxetine), but not with two tricyclic antidepressants (imipramine, clomipramine) and was abolished by pre-treatment with para-chlorophenylalanine, an inhibitor of serotonin synthesis. Using microdialysis experiments, we demonstrated that NOP-receptor activation by infusion of nociceptin 10(-6) M or 10(-5) M increased the level of extracellular serotonin in the dorsal raphe nucleus. This effect was abolished by co-infusion of the NOP-receptor antagonist UFP 101. These results confirm the existence of reciprocal interactions between serotonin and nociceptin/NOP transmissions in the dorsal raphe nucleus.

Effect of potent endomorphin degradation blockers on analgesic and antidepressant-like responses in mice.

The biological effects of endomorphins (EMs) are short-lasting due to their rapid degradation by endogenous enzymes. Competing enzymatic degradation is an approach to prolong EM bioavailability. In the present study, a series of tetra- and tripeptides of similar to EMs structure was synthesized and tested in vitro and in vivo for their ability to inhibit degradation of EMs. The obtained results indicated that, among the series of analogs, the tetrapeptide Tyr-Pro-d-ClPhe-Phe-NH(2) and the tripeptide Tyr-Pro-Ala-NH(2), which did not bind to the µ-opioid receptors, were potent inhibitors of EM catabolism in rat brain homogenate. In vivo, these two peptides significantly prolonged the analgesic and antidepressant-like effects, induced by exogenous EMs, by blocking EM degrading enzymes. These new potent inhibitors may therefore increase the level and the half life of endogenous EMs and could be used in a new therapeutic strategy against pain and mood disorders, based on increasing of EM bioavailability.

Synthesis of pyrazolines by a site isolated resin-bound reagents methodology.

The elaboration of biologically important 3,4-substituted pyrazolines was achieved by an organocatalysed aza-Michael/transimination domino sequence between hydrazones and enones making use of a mixture of heterogeneous resin-bound acid/base reagents. This methodology nicely illustrates the site isolation concept of supported reagents allowing the simultaneous use of otherwise destructive reactive functionalities.

Novel endomorphin analogues with antagonist activity at the mu-opioid receptor in the gastrointestinal tract.

Opioid bowel dysfunction (OBD) summarizes common adverse side effects of opiate-based management of pain. A promising therapeutic approach to prevent OBD and other opioid-related disorders of the gastrointestinal (GI) tract is the co-administration of opiates with peripherally-restricted mu-opioid receptor (MOR)-selective antagonists. The aim of this study was to investigate the selectivity and efficacy of three novel peptide antagonists: antanal-1, antanal-2, and antanal-2A at MOR in the GI tract in vitro and in vivo. The effects of the antanals on GI motility were studied in vitro, using isolated preparations of mouse ileum and colon and in vivo, by measuring colonic propulsion in mice. Additionally, in vitro stability against enzymatic degradation and blood-brain barrier (BBB) permeability using the hot plate test in mice were examined. The antanals significantly reduced the inhibitory effect of the MOR agonists endomorphin-2, morphine, and loperamide on mouse ileum and colon contractions in vitro and blocked morphine-induced decrease of colonic bead expulsion in vivo. The hot plate test in mice showed that the antagonist activity of all antanals was restricted to the periphery. Antanal-1, antanal-2, and antanal-2A are promising MOR antagonists with limited BBB permeability, which may be developed into future therapeutics of opioid-related GI dysfunction.

Toxicity of a treatment associating dopamine and disulfiram for catecholaminergic neuroblastoma SH-SY5Y cells: relationships with 3,4-dihydroxyphenylacetaldehyde formation.

3,4-Dihydroxyphenylacetaldehyde (DOPAL) is formed by the oxidative deamination of dopamine (DA) catalyzed by monoamine oxidases (MAO); then, the aldehyde is oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenases (ALDH) or reduced to 3,4-dihydroxyphenylethanol (DOPET) by aldose/aldehyde reductases. The present work aimed at evaluating the in vitro toxicity of DOPAL on catecholaminergic neuroblastoma SH-SY5Y cells which accumulate DA. DOPAL synthesis was stimulated by incubating cells with DA and blocking DOPAL oxidation by disulfiram, an irreversible inhibitor of ALDH. As evidenced by MTT reduction assays, DA and disulfiram treatments produced cell losses which increased with time. 10(-2)M DA reduced by 40% cell viability after a 1h treatment, when its TC(50) (concentration reducing viability by 50%) value was 7.3 x 10(-5) M after a 24 h treatment. For the same treatment periods, TC(50) values for disulfiram were 8 x 10(-5) and 8.7 x 10 (-7) M, respectively. MTT reduction assay performed after a 24h treatment followed by a 24h incubation in a drug-free medium evidenced that the toxicity of 10(-4)M DA or 10(-6)M disulfiram was potentiated by the second drug. HPLC measurements showed that DOPAL was produced at the early stages of the treatment by DA and disulfiram. This was evidenced by the significant increase in the ((DOPAL + DOPET)/DOPAC ratio observed after a combined 3h treatment by 10(-4)M DA and 10(-6)M disulfiram. Total contents in DA and DOPAL were greatly reduced at the end of a 15 h treatment, and disulfiram did not significantly enhanced the (DOPAL + DOPET)/DOPAC ratio. For both treatment durations, DOPAL and DOPET were detectable only in the extracellular medium. So, these results suggest that an early production of DOPAL could produce delayed toxic effects on SH-SY5Y cells. Production of DOPET and release of DOPAL could be important means for reducing DOPAL concentrations in dopaminergic neurons.

Semi-chronic increase in striatal level of 3,4-dihydroxyphenylacetaldehyde does not result in alteration of nigrostriatal dopaminergic neurones.

This work was carried out to evaluate the potential in vivo toxicity of 3,4-dihydroxyphenylacetaldehyde (DOPAL), an aldehyde formed from dopamine by monoamine oxidase (MAO) that is oxidised mainly to 3,4-dihydroxyphenylacetic acid (DOPAC) by brain aldehyde dehydrogenases (ALDH). In this study, male Sprague-Dawley rats were treated with levodopa (L-dopa)-benserazide, which increases DOPAL production by MAO, and disulfiram, an irreversible inhibitor of ALDH, which reduces the formation of DOPAC from DOPAL. An acute systemic intraperitoneal (i.p.) injection of 100 mg/kg disulfiram and L-dopa-benserazide (100 mg/kg + 25 mg/kg, 24 hr later) significantly increased DOPAL striatal level. A 30-day treatment with disulfiram (100 mg/kg i.p., once every 2 days) and L-dopa-benserazide (100 mg/kg + 25 mg/kg, two times/day) did not affect either indexes used to assess integrity of the nigrostriatal dopaminergic neurones (i.e., the striatal content in dopamine and binding to the vesicular monoamine transporter on striatal membranes). These results do not evidence any deleterious effect of DOPAL and argue against toxicity of L-dopa therapy.

Interactions of cations and anions with the binding of uptake blockers to the dopamine transporter.

Uptake blockers and substrates are likely to recognise a common binding domain on the dopamine neuronal transporter (DAT). Among cations that form ionic gradients at the level of the cellular plasma membrane, Na+ is the only one that can stimulate their binding. The binding stimulation appears over Na+ concentrations ranging from 0 to 10-60 mM; at higher Na+ concentrations, binding reaches a plateau or decreases, according to the uptake blocker that is studied. The majority of the other cations, including K+, Ca2+, Mg2+ and Tris+, inhibit the binding of uptake blockers. Several metals impair binding to the DAT and/or the dopamine transport, but, under specific conditions, some of them, and chiefly Zn2+, stimulate binding. The complex relationships between cations, uptake blockers and the DAT suggest that cations recognise at least three different sites: the first one, site 1, is for cation-induced binding inhibition; the second one, site 2, is for Na+-induced binding stimulation; and the third one, site 3, is for Zn2+-induced binding stimulation. Modelling of the interactions between Na+, K+ and radioligands allows a better understanding of the effects of cations at sites 1 and 2, and of uptake blockers at site 1. Some anions also facilitate the binding of uptake blockers to the DAT, as far as they are associated with Na+. The dependence of the binding of dopamine on ions could be involved in its preferential inward transport and used by uptake blockers for their own binding to the DAT.