Therapeutic antidepressant potential of a conjugated siRNA silencing the serotonin transporter after intranasal administration.

Major depression brings about a heavy socio-economic burden worldwide due to its high prevalence and the low efficacy of antidepressant drugs, mostly inhibiting the serotonin transporter (SERT). As a result, ~80% of patients show recurrent or chronic depression, resulting in a poor quality of life and increased suicide risk. RNA interference (RNAi) strategies have been preliminarily used to evoke antidepressant-like responses in experimental animals. However, the main limitation for the medical use of RNAi is the extreme difficulty to deliver oligonucleotides to selected neurons/systems in the mammalian brain. Here we show that the intranasal administration of a sertraline-conjugated small interfering RNA (C-SERT-siRNA) silenced SERT expression/function and evoked fast antidepressant-like responses in mice. After crossing the permeable olfactory epithelium, the sertraline-conjugated-siRNA was internalized and transported to serotonin cell bodies by deep Rab-7-associated endomembrane vesicles. Seven-day C-SERT-siRNA evoked similar or more marked responses than 28-day fluoxetine treatment. Hence, C-SERT-siRNA (i) downregulated 5-HT1A-autoreceptors and facilitated forebrain serotonin neurotransmission, (ii) accelerated the proliferation of neuronal precursors and (iii) increased hippocampal complexity and plasticity. Further, short-term C-SERT-siRNA reversed depressive-like behaviors in corticosterone-treated mice. The present results show the feasibility of evoking antidepressant-like responses by selectively targeting neuronal populations with appropriate siRNA strategies, opening a way for further translational studies.

RNAi-mediated serotonin transporter suppression rapidly increases serotonergic neurotransmission and hippocampal neurogenesis.

Current antidepressants, which inhibit the serotonin transporter (SERT), display limited efficacy and slow onset of action. Here, we show that partial reduction of SERT expression by small interference RNA (SERT-siRNA) decreased immobility in the tail suspension test, displaying an antidepressant potential. Moreover, short-term SERT-siRNA treatment modified mouse brain variables considered to be key markers of antidepressant action: reduced expression and function of 5-HT(1A)-autoreceptors, elevated extracellular serotonin in forebrain and increased neurogenesis and expression of plasticity-related genes (BDNF, VEGF, Arc) in hippocampus. Remarkably, these effects occurred much earlier and were of greater magnitude than those evoked by long-term fluoxetine treatment. These findings highlight the critical role of SERT in serotonergic function and show that the reduction of SERT expression regulates serotonergic neurotransmission more potently than pharmacological blockade of SERT. The use of siRNA-targeting genes in serotonin neurons (SERT, 5-HT(1A)-autoreceptor) may be a novel therapeutic strategy to develop fast-acting antidepressants.

Reduced signal transduction by 5-HT4 receptors after long-term venlafaxine treatment in rats.

BACKGROUND AND PURPOSE: The 5-HT(4) receptor may be a target for antidepressant drugs. Here we have examined the effects of the dual antidepressant, venlafaxine, on 5-HT(4) receptor-mediated signalling events. EXPERIMENTAL APPROACH: The effects of 21 days treatment (p.o.) with high (40 mg·kg(-1)) and low (10 mg·kg(-1)) doses of venlafaxine, were evaluated at different levels of 5-HT(4) receptor-mediated neurotransmission by using in situ hybridization, receptor autoradiography, adenylate cyclase assays and electrophysiological recordings in rat brain. The selective noradrenaline reuptake inhibitor, reboxetine (10 mg·kg(-1), 21 days) was also evaluated on 5-HT(4) receptor density. KEY RESULTS: Treatment with a high dose (40 mg·kg(-1)) of venlafaxine did not alter 5-HT(4) mRNA expression, but decreased the density of 5-HT(4) receptors in caudate-putamen (% reduction = 26 ± 6), hippocampus (% reduction = 39 ± 7 and 39 ± 8 for CA1 and CA3 respectively) and substantia nigra (% reduction = 49 ± 5). Zacopride-stimulated adenylate cyclase activation was unaltered following low-dose treatment (10 mg·kg(-1)) while it was attenuated in rats treated with 40 mg·kg(-1) of venlafaxine (% reduction = 51 ± 2). Furthermore, the amplitude of population spike in pyramidal cells of CA1 of hippocampus induced by zacopride was significantly attenuated in rats receiving either dose of venlafaxine. Chronic reboxetine did not modify 5-HT(4) receptor density. CONCLUSIONS AND IMPLICATIONS: Our data indicate a functional desensitization of 5-HT(4) receptors after chronic venlafaxine, similar to that observed after treatment with the classical selective inhibitors of 5-HT reuptake.

Ion channels and epilepsy.

The role of voltage-gated and ligand-gated ion channels in epileptogenesis of both genetic and acquired epilepsies, and as targets in the development of new antiepileptic drugs (AEDs) is reviewed. Voltage-gated Na+ channels are essential for action potentials, and their mutations are the substrate for generalised epilepsy with febrile seizures plus and benign familial neonatal infantile seizures; Na+ channel inhibition is the primary mechanism of carbamazepine, phenytoin and lamotrigine, and is a probable mechanism for many other classic and novel AEDs. Voltage-gated K+ channels are essential in the repolarisation and hyperpolarisation that follows paroxysmal depolarisation shifts (PDSs), and their mutations are the substrate for the benign neonatal epilepsy and episodic ataxia type 1; they are new targets for AEDs such as retigabine. Voltage-gated Ca2+ channels are involved in neurotransmitter release, in the sustained depolarisation-phase of PDSs, and in the generation of absence seizures; their mutations are a substrate for juvenile myoclonic epilepsy and the absence-like pattern seen in some mice; the antiabsence effect of ethosuximide is due to the inhibition of thalamic T-type Ca2+ channels. Voltage-gated Cl- channels are implicated in GABA(A) transmission, and mutations in these channels have been described in some families with juvenile myoclonic epilepsies, epilepsy with grand mal seizures on awakening or juvenile absence epilepsy. Hyperpolarisation-activated cation channels have been implicated in spike-wave seizures and in hippocampal epileptiform discharges. The Cl- ionophore of the GABA(A) receptor is responsible for the rapid post-PDS hyperpolarisation, it has been involved in epileptogenesis both in animals and humans, and mutations in these receptors have been found in families with juvenile myoclonic epilepsy or generalised epilepsy with febrile seizures plus; enhancement of GABA(A) inhibitory transmission is the primary mechanism of benzodiazepines and phenobarbital and is a mechanistic approach to the development of novel AEDs such as tiagabine or vigabatrin. Altered GABA(B)-receptor function is implicated in spike-wave seizures. Ionotropic glutamate receptors are implicated in the sustained depolarisation phase of PDS and in epileptogenesis both in animals and humans; felbamate, phenobarbital and topiramate block these receptors, and attenuation of glutamatergic excitatory transmission is another new mechanistic approach. Mutations in the nicotinic acetylcholine receptor are the substrates for the nocturnal frontal lobe epilepsy. The knowledge of the role of the ion channels in the epilepsies is allowing the design of new and more specific therapeutic strategies.

[Advances in the physiopathology of epileptogenesis: molecular aspects]. / Avances en la fisiopatología de la epileptogénesis: aspectos moleculares.

OBJECTIVE: We review the molecular basis of epileptogenesis and the new perspectives in the treatment of epilepsy. DEVELOPMENT: Epileptogenesis are the molecular and cellular events producing the disordered firing of a subpopulation of neurons resulting in periodic seizures. Epilepsies may be due to genetic and acquired factors. Some idiopathic epilepsies are due to mutant genes coding voltage gated sodium and potassium channels, GABAA receptor chloride channels and nicotinic acetylcholine receptor sodium channels. Genetic defects also produce epilepsy secondary to either neuronal developmental or metabolic abnormalities, and may contribute to acquired epilepsy. Events observed in both animal and human acquired epilepsies are an increase in glutamate levels and NMDA receptor sensitivity, selective lost of pyramidal neurons, mossy fibre sprouting and neosinaptogenesis. There is also a reduction in inhibitory control due to lost of GABAergic interneurons, and a decrease in GABA levels and GABAA receptor sensitivity. Hyperexcitability may be also due to reduction in glial ATPasa activity, increase in astrocytes gap junctions, and decrease in extracellular calcium. Chandelier GABAergic interneuron microlesions and an hyperexcitable thalamus are key in spread of partial seizures. Absences may be caused by cortex hyperexcitability and genetic abnormalities in thalamic voltage gated T calcium channels. Brain stem is key in convulsive seizures. The role of voltage gated potassium, sodium and calcium channels, and GABAergic and glutamatergic neurotransmission in epileptogenesis and treatment of epilepsies are revised. The role of other neurotransmitters and neuromodulators, second messengers, and immediate early genes and neurotrophins are also commented. CONCLUSION: Understanding the molecular basis of epileptogenesis should lead to the rational design of drugs both to prevent the development of epilepsy, and minimize hyperexcitability which may be the result of a genetic or acquired disorder.

Involvement of the cyclic AMP system in the switch from tolerance into supersensitivity to the antinociceptive effect of the opioid sufentanil.

1. We have previously demonstrated that chronic and simultaneous treatment of rats with the mu-opioid receptor agonist sufentanil and the Ca(2+) channel blocker nimodipine, not only prevented tolerance development, but the animals became supersensitive to the antinociceptive effect of the opioid. The focus of the present work was to determine the possible involvement of cross interactions between the adenylyl cyclase pathway and L-type voltage-sensitive Ca(2+)-channels, in modulating the switch from opioid tolerance into supersensitivity. 2. The modulatory effect of sufentanil on adenylyl cyclase activity was determined by measuring cyclic AMP production in slices from the cortex of rats rendered tolerant or supersensitive to the antinociceptive effect of the opioid. Tolerance was induced by chronic infusion of sufentanil, at a rate of 2 microg h(-1), for 7 days. Supersensitivity was induced by concurrent infusion of sufentanil (2 microg h(-1)) and nimodipine (1 microg h(-1)) for 7 days. Antinociception was evaluated by the tail-flick test. 3. Tolerance to the analgesic effect of sufentanil was associated with a significant reduction in the response of adenylyl cyclase to forskolin. Furthermore, the effect of the opioid on forskolin-induced cyclic AMP accumulation was abolished. On the other hand, supersensitivity to the analgesic effect of the opioid was associated with an increase in both, the adenylyl cyclase response to forskolin, and the opioid inhibition of cyclic AMP production. 4. We suggest that sustained L-type Ca(2+) channel blockade may result in changes in the adenylyl cyclase effector system triggered by mu-opioid receptor activation, leading to the switch from opioid tolerance into supersensitivity.

Time course of the GABAergic effects of vigabatrin: is the time course of brain GABA related to platelet GABA-transaminase inhibition?

PURPOSE: To analyze the time course of the effects of vigabatrin (VGB) on brain gamma-aminobutyric acid (GABA), and its relation with 4-aminobutyrate-2-ketoglutarate amino-transferase (GABA-T) in brain and platelets. METHODS: Blood and brain samples were collected at 4, 24, 48, and 72 h after a single dose and after 3 and 8 days of treatment with 200 mg/kg of VGB in rats. RESULTS: Time courses of the GABAergic effects of VGB were different after single and multiple doses: with multiple doses, the inhibition of brain GABA-T was quicker and longer, the inhibition of platelet GABA-T was greater and longer, the increase in brain GABA was greater, and recovery began earlier. After pooling the data obtained at 4, 24, 48, and 72 h, we observed a power correlation between the increase in brain GABA in individual rats as percentage of the control and both the inhibition of brain GABA-T after a single dose of VGB (r = -0.40; p < 0.05), and the inhibition of platelet GABA-T after 3 days (r = -0.48; p < 0.01) and 8 days of treatment (r = -0.53; p < 0.01). When all data after single and multiple doses were pooled, the increase in brain GABA correlated better with the inhibition of GABA-T in platelets (r = -0.62; p < 0.001) than in brain (r = -0.38; p < 0.001). Platelet GABA-T correlated with brain GABA at 4 h (r = -0.64; p < 0.001) and 24 h (r = -0.66; p < 0.001) but not at 48 and 72 h. CONCLUSIONS: Platelet GABA-T reflects the time course of the increase in brain GABA better than does brain GABA-T after multiple doses of VGB in rats.

Effects of increasing doses of vigabatrin on platelet gamma-aminobutyric acid-transaminase and brain gamma-aminobutyric acid in rats.

We report the relationship of GABA-transaminase inhibition in platelets and brain with the increase in brain gamma-aminobutyric acid (GABA), as percents of the control, at 24 h after single and after 3 and 8 days of treatment with increasing doses (1, 3, 10, 30, 100 and 300 mg kg(-1) day(-1) of vigabatrin in rats. The inhibition of GABA-transaminase in platelets correlated at least as well as that in brain with the increase in brain GABA after 3 days (r = - 0.87 vs. r = -0.78), and 8 days of treatment (r = -0.77 vs. r = -0.74), and when the data of single and multiple doses were pooled (r = -0.77 vs. r = -0.75). The correlation between platelet GABA-transaminase and brain GABA fitted to a power curve, the increase in brain GABA being significant only when platelet GABA-transaminase was inhibited to less than 50% of the control. Our results suggest that platelet GABA-transaminase could be a peripheral marker of the effect of vigabatrin on brain GABA in rats.

Induction of toxin sensitivity in insect cells by infection with baculovirus encoding diphtheria toxin receptor.

The diphtheria toxin receptor (DTR) has been identified as the precursor of heparin-binding epidermal growth factor-like growth factor, which may interact with other membrane proteins to form the functional receptor. To test if mammalian DTR is able to confer toxin sensitivity onto phylogenetically distant cells, we expressed monkey DTR in the baculovirus system and tested infected insect cells for toxin sensitivity. cDNA encoding an epitope-tagged heparin-binding epidermal growth factor-like growth factor precursor (DTRB3) was inserted into the virus genome by allelic replacement to construct the recombinant virus vAc-DTRB3. SF9 cells infected with vAc-DTRB3 expressed functional DTR, which could be precipitated from the solubilized membrane fraction of infected cells with Sepharose-immobilized diphtheria toxin. The highest level of expression (about 5 x 10(6) receptors/cell) was observed 48 h after infection, at which time the infected cells were highly sensitive to diphtheria toxin. Uninfected SF9 cells and cells infected with the wild type virus were resistant to the toxin. The presence of heparin increased both the binding and the toxin sensitivity of vAc-DTRB3-infected SF9 cells. Translocation of toxin A fragment was induced when cells with surface-bound toxin were exposed to low pH, and the translocation was optimal at pH < or = 5.5. It was approximately 100 times more efficient at 24 degrees C than at 4 degrees C. The data indicate that monkey DTR is fully functional when expressed in insect cells.

Coadministration of vigabatrin and valproate in children with refractory epilepsy.

The effects of adding vigabatrin (GVG) to the antiepileptic regimens of 16 children with refractory epilepsy have been studied. One-half of the regimens included sodium valproate (VPA). Parameters studied were seizure reduction, platelet GABA-T activity, and steady-state plasma concentrations (CSS) of GVG and VPA. Add-on GVG reduced the seizure frequency both in patients receiving VPA (from 42.9 to 4.5 seizures/month, p < 0.01) and in those without VPA (from 60.0 to 31.7 seizures/month, p < 0.05). GVG also reduced GABA-T activity in both groups (from 19.4 to 5.4, p < 0.001 and from 8.3 to 4.5 pmol/min/mg of protein, p < 0.05, respectively). Seizure reduction and GABA-T inhibition were greater in patients taking VPA than in those who were not. In patients receiving VPA, no significant changes were observed in VPA CSS values before and after the addition of GVG. On the other hand, no differences were found in GVG CSS values between patients with and without VPA. It is concluded that the coadministration of GVG to valproate reduces the frequency of seizures in refractory epileptic children and does not affect the steady-state plasma concentrations of either drug. Therefore, their association could be useful in clinical practice.

Gamma-vinyl GABA (vigabatrin): relationship between dosage, plasma concentrations, platelet GABA-transaminase inhibition, and seizure reduction in epileptic children.

The relationship between vigabatrin gamma-vinyl GABA (GVG, vigabatrin) daily dosage or steady-state plasma concentrations (CSS), platelet GABA-transaminase (GABA-T) inhibition, and seizure reduction were studied in 16 children with refractory epilepsy. After 2 months of observation and 1 month of single-blind add-on placebo, a fixed GVG dosage was added for 2 months. The dosage was then adjusted in two 2-month periods each, based on the patient's clinical response. In the fixed-dose period, GVG dosages of 56.8 mg/kg/day and CSS of 8.1 mg/L reduced GABA-T activity from 13.9 to 5.1 pmol/min/mg protein (p less than 0.001) and that of seizures from 51.4 to 22.3 seizures per month (p less than 0.01). Seizure reduction was correlated with dosage (r = 0.83, p less than 0.001), but not with CSS or with platelet GABA-T inhibition. After the GVG dose-adjustment periods, in which dosages of 84.4 mg/kg/day and CSS of 10.6 mg/L were reached, only a slight reduction was observed in both GABA-T activity (from 5.1 to 4.9 pmol/min/mg protein) and seizures (from 22.3 to 18.1 seizures per month). In GVG-responsive patients (excluding placebo-sensitive and GVG-resistant patients), a greater reduction of seizures was achieved (from 17.0 to 7.1 seizures per month, p less than 0.05), which was not accompanied by greater inhibition of GABA-T. GVG treatment in children should be started with a dosage of 50 mg/kg/day, increased to 75 or even 100 mg/kg/day when a partial response is observed. If seizures do not improve or if they become worse, the patient should be considered resistant and GVG should be discontinued.

Effects of single and multiple increasing doses of vigabatrin on brain GABA metabolism and correlation with vigabatrin plasma concentration.

UNLABELLED: The effects of increasing (50-1600 mg/kg/day) doses of vigabatrin (GVG) both as single doses and after 8 or 28 days of treatment have been studied in 19 groups of 10 adult Wistar rats. The parameters studied were brain gamma-aminobutyric acid-transaminase (GABA-T) activity, GABA concentration and L-glutamate decarboxylase (GAD) activity. Single increasing doses of GVG progressively inhibited GABA-T activity, but a residual activity of about 40% was observed with the highest doses. GABA concentration increased in a dose-dependent manner but a ceiling was not reached. GAD activity was slightly inhibited at low doses and stimulated at high ones. When treatment was continued for 8 days, more marked effects of GVG on GABA-T and GABA, a more severe toxicity and higher GVG plasma concentrations were observed. GAD was inhibited instead of stimulated by high GVG doses. After 28 days of treatment the effects of GVG on GABA-T and GABA were similar to those after 8 days. However, toxic effects decreased and lower GVG plasma concentrations were found. IN CONCLUSION: (a) the more marked brain GABAergic effects observed after 8 days of treatment with GVG may explain the greater anticonvulsant effects observed by others in animals, and (b) GVG plasma concentrations correlate well with changes in brain GABA-T and GABA, and may partly explain changes in the effects of GVG related to the length of treatment.

Relationship between platelet and brain GABA transaminase inhibition by single and multiple doses of vigabatrin in rats.

Vigabatrin (gamma-vinyl-GABA, GVG) is an inhibitor of brain GABA transaminase (GABA-T) that also inhibits platelet GABA-T in rats and humans. We have compared the effects of single and multiple doses of GVG on both enzymes in 19 groups of 10 adult male Wistar rats, treated with increasing GVG doses (0-1,600 mg/kg/day) for 1, 8, and 28 days. The platelet GABA-T was more sensitive to the inhibitory effects of GVG than the brain enzyme was especially with low dosages of GVG. After 8 days of treatment, higher GVG plasma levels and a higher inhibition of both enzymes were shown. However, after 28 days, lower GVG plasma levels and similar inhibition of both enzymes compared to the eighth day were found. Correlations between platelet and brain GABA-T for individual rats were statistically significant after 1 day (r = 0.40, p less than 0.01) but not after 8 and 28 days of treatment because of the total inhibition of platelet GABA-T and only partial inhibition of brain GABA-T. We concluded the following: (a) platelet GABA-T is more inhibited than brain GABA-T when low doses of GVG are used and (b) multiple doses reach a higher inhibition of both enzymes than single doses, which could be explained by an increase in GVG concentrations.