First evidence of protective effects on stroke recovery and post-stroke depression induced by sortilin-derived peptides.

Post-stroke depression (PSD) is the most common mood disorder following stroke with high relevance for outcome and survival of patients. The TREK-1 channel represents a crucial target in the pathogenesis of stroke and depression. Spadin and its short analog mini-spadin were reported to display potent antidepressant properties. We investigated the therapeutic effects of mini-spadin in a mouse model of focal ischemia and PSD. To activate TREK-1 and induce neuroprotection a single low dose of mini-spadin (0.03 µg/kg) was intraperitoneally injected 30  min after the onset of ischemia, once a day during 7 days post-ischemia. Then, to inhibit TREK-1 and induce antidepressant effect, the peptide was injected at higher concentration (3 µg/kg) once a day for 4 days/week until the sacrifice of animals. Electrophysiological studies showed that mini-spadin had a biphasic action on TREK-1. At low doses, the channel activity was increased whereas at higher doses it was inhibited. Mini-spadin prevented the loss of body weight and the delayed dopaminergic degeneration in substantia nigra and improved the motor and cognitive ischemia-induced deficits. Moreover, mini-spadin prevented PSD analyzed in the Forced Swim (FST) and Novelty Suppressed Feeding (NSF) tests. Finally, enhanced neurogenesis and synaptogenesis contributed to the beneficial effects of mini-spadin against stroke and PSD. This work reveals the first evidence that the modulation of TREK-1 channels in the early and chronic phases of stroke as well as the stimulation of brain plasticity by mini-spadin could play a key role in its brain protective effects against stroke and its deleterious consequences such as PSD.

Role of TREK-1 in Health and Disease, Focus on the Central Nervous System.

TREK-1 is the most studied background K2P channel. Its main role is to control cell excitability and maintain the membrane potential below the threshold of depolarization. TREK-1 is multi-regulated by a variety of physical and chemical stimuli which makes it a very promising and challenging target in the treatment of several pathologies. It is mainly expressed in the brain but also in heart, smooth muscle cells, endocrine pancreas, and prostate. In the nervous system, TREK-1 is involved in many physiological and pathological processes such as depression, neuroprotection, pain, and anesthesia. These properties explain why many laboratories and pharmaceutical companies have been focusing their research on screening and developing highly efficient modulators of TREK-1 channels. In this review, we summarize the different roles of TREK-1 that have been investigated so far in attempt to characterize pharmacological tools and new molecules to modulate cellular functions controlled by TREK-1.

Fighting against depression with TREK-1 blockers: Past and future. A focus on spadin.

Depression is a devastating mood disorder and a leading cause of disability worldwide. Depression affects approximately one in five individuals in the world and represents heavy economic and social burdens. The neurobiological mechanisms of depression are not fully understood, but evidence highlights the role of monoamine neurotransmitter balance. Several antidepressants (ADs) are marketed to treat depression and related mood disorders. However, despite their efficacy, they remain nonspecific and unsafe because they trigger serious adverse effects. Therefore, developing new molecules for new targets in depression has become a real necessity. Eight years ago, spadin was described as a natural peptide with AD properties. This 17-amino acid peptide blocks TREK-1 channels, an original target in depression. Compared to the classical AD drugs such as fluoxetine, which requires 3-4 weeks for the AD effect to manifest, spadin acts rapidly within only 4 days of treatment. The AD properties are associated with increased neurogenesis and synaptogenesis in the brain. Despite the advantages of this fast-acting AD, the in vivo stability is weak and does not last for >7 h. The present review summarizes different strategies such as retro-inverso strategy, cyclization, and shortening the spadin sequence that has led to the development and optimization of spadin as an AD. Shortened spadin analogs present increased inhibition potency for TREK-1, an improved AD activity, and prolonged in vivo bioavailability. Finally, we also discuss about other inhibitors of TREK-1 channels with a proven efficacy in treating depression in the clinic, such as fluoxetine.

Identification and characterization of two zebrafish Twik related potassium channels, Kcnk2a and Kcnk2b.

KCNK2 is a 2 pore domain potassium channel involved in maintaining cellular membrane resting potentials. Although KCNK2 is regarded as a mechanosensitive ion channel, it can also be gated chemically. Previous research indicates that KCNK2 expression is particularly enriched in neuronal and cardiac tissues. In this respect, KCNK2 plays an important role in neuroprotection and has also been linked to cardiac arrhythmias. KCNK2 has subsequently become an attractive pharmacologic target for developing preventative/curative strategies for neuro/cardio pathophysiological conditions. Zebrafish represent an important in vivo model for rapidly analysing pharmacological compounds. We therefore sought to identify and characterise zebrafish kcnk2 to allow this model system to be incorporated into therapeutic research. Our data indicates that zebrafish possess two kcnk2 orthologs, kcnk2a and kcnk2b. Electrophysiological analysis of both zebrafish Kcnk2 orthologs shows that, like their human counterparts, they are activated by different physiological stimuli such as mechanical stretch, polyunsaturated fatty acids and intracellular acidification. Furthermore, both zebrafish Kcnk2 channels are inhibited by the human KCNK2 inhibitory peptide spadin. Taken together, our results demonstrate that both Kcnk2a and Kcnk2b share similar biophysiological and pharmacological properties to human KCNK2 and indicate that the zebrafish will be a useful model for developing KCNK2 targeting strategies.

Increased serum levels of sortilin-derived propeptide after electroconvulsive therapy in treatment-resistant depressed patients.

PURPOSE: Sortilin-derived propeptide (PE) and its synthetic analog spadin show strong antidepressant activity in rodents and, therefore, could be used as a biomarker to evaluate the clinical efficacy of antidepressant treatments. The aim of this study was to determine whether electroconvulsive therapy (ECT) modulates serum PE concentration in patients with treatment-resistant depression (TRD). PATIENTS AND METHODS: Forty-five patients with major depressive disorder, who met the Diagnostic and Statistical Manual of Mental Disorders-IV criteria, were selected for this study. RESULTS: We did not observe any difference in the PE levels between TRD patients and controls (z=0.10, P=0.92), but we found a strong significant increase between the PE levels measured just before (T0) and about 1 month (T2) after ECT (z=-2.82, P=0.005). A significant difference between T0 and T2 was observed only in responders (z=-2.59, P=0.01), whereas no effect was found in nonresponders (z=-1.27, P=0.20). Interestingly, we found a significant correlation between the increase in PE levels and decrease in Montgomery -Åsberg Depression Rating Scale scores for the total patient sample (P=0.03). CONCLUSION: This study indicates for the first time that ECT affects serum PE concentration in responders and, therefore, could contribute to the evaluation of the therapy success.

Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior.

The background potassium channel TREK-1 has been shown to be a potent target for depression treatment. Indeed, deletion of this channel in mice resulted in a depression resistant phenotype. The association of TREK-1 with the sorting protein sortilin prompted us to investigate the behavior of mice deleted from the gene encoding sortilin (Sort1-/-). To characterize the consequences of sortilin deletion on TREK-1 activity, we combined behavioral, electrophysiological and biochemical approaches performed in vivo and in vitro. Analyses of Sort1-/- mice revealed that they display: (1) a corticosterone-independent anxiety-like behavior, (2) a resistance to depression as demonstrated by several behavioral tests, and (3) an increased activity of dorsal raphe nucleus neurons. All these properties were associated with TREK-1 action deficiency consequently to a decrease of its cell surface expression and to the modification of its electrophysiological activity. An increase of BDNF expression through activation of the furin-dependent constitutive pathway as well as an increase of the activated BDNF receptor TrkB were in agreement with the decrease of depressive-like behavior of Sort1-/- mice. Our results demonstrate that the TREK-1 expression and function are altered in the absence of sortilin confirming the importance of this channel in the regulation on the mood as a crucial target to treat depression.

The Involvement of Sortilin/NTSR3 in Depression as the Progenitor of Spadin and Its Role in the Membrane Expression of TREK-1.

The molecular identification of sortilin, also called neurotensin receptor-3, from three different biochemical approaches already predicted the involvement of the protein in numerous biological and cellular functions. The first important observation was that sortilin is synthesized as a precursor that is converted to a mature protein after cleavage by the protein convertase furin in late Golgi compartments. This maturation leads to the formation of a 44 amino acid peptide, the propeptide (PE). The release of this peptide when matured sortilin reached the plasma membrane remained to be demonstrated. Sortilin has been also shown to be shedded by matrix metalloproteases releasing a large extracellular fragment identified as soluble sortilin. Therefore, sortilin has been shown to interact with several proteins and receptors confirming its role in the sorting of cellular components to the plasma membrane and/or to the lysosomal pathway. Interestingly, sortilin physically interacts with the two pore domain potassium channel TREK-1 and the PE as well as its synthetic analog spadin is able to block the activation of TREK-1 highlighting their role in the depression pathology. The present review describes the advance of research that led to these results and how both the soluble form of sortilin and the sortilin-derived PE have been detected in human serum and whose levels are affected in patients with major depressive disorder (MDD). The use of spadin as an antidepressant and the further role of soluble sortilin and of sortilin-derived PE as potential biomarkers during depression statement and/or remission of the pathology are considered and discussed in this review.

Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity.

Depression is a devastating mental disorder that affects 20% of the population worldwide. Despite their proven efficacy, antidepressants present a delayed onset of action and serious adverse effects. Seven years ago, we described spadin (PE 12-28) as a promising endogenous peptide with antidepressant activity. Spadin specifically blocks the TREK-1 channel. Previously, we showed in vivo that, spadin activity disappeared beyond 7 h after administration. In order to improve in vivo spadin stability and bioavailability, we screened spadin analogs and derivatives. From the study of spadin blood degradation products, we designed a 7 amino-acid peptide, PE 22-28. In vitro studies on hTREK-1/HEK cells by using patch-clamp technique, showed that PE 22-28 displayed a better specificity and affinity for TREK-1 channel compared to spadin, IC50 of 0.12 nM vs. 40-60 nM for spadin. In the same conditions, we also pointed out that different modifications of its N or C-terminal ends maintained or abolished TREK-1 channel activity without affecting PE 22-28 affinity. In vivo, the antidepressant properties of PE 22-28 and its derivatives were demonstrated in behavioral models of depression, such as the forced swimming test. Mice treated with spadin-analogs showed a significant reduction of the immobility time. Moreover, in the novelty suppressed feeding test after a 4-day sub-chronic treatment PE 22-28 reduced significantly the latency to eat the food pellet. PE 22-28 and its analogs were able to induce neurogenesis after only a 4-day treatment with a prominent effect of the G/A-PE 22-28. On mouse cortical neurons, PE 22-28 and its derivatives enhanced synaptogenesis measured by the increase of PSD-95 expression level. Finally, the action duration of PE 22-28 and its analogs was largely improved in comparison with that of spadin, up to 23 h instead of 7 h. Taken together, our results demonstrated that PE 22-28 and its derivatives represent other promising molecules that could be an alternative to spadin in the treatment of depression.

Serum sortilin-derived propeptides concentrations are decreased in major depressive disorder patients.

BACKGROUND: Despite intense research on mechanisms underlying the depressive pathophysiology, reliable biomarkers to assess antidepressant treatment response are still lacking. Since the sortilin-derived propeptide (PE) displays potent antidepressant activities and can be measured in the blood of rodents, we wondered whether in human its seric level can vary between patients affected by major depressive disorder (MDD) and healthy controls and after antidepressant treatment. METHODS: By using a specific dosing method, characterized by structure-recognition analysis with various synthesized PE analogues, we conducted a translational study to test whether blood levels of PE are under pathophysiological regulation and could serve as biomarkers of the depression state. RESULTS: The serum concentration of PE, a peptide displaying potent antidepressant activities in rodents, is decreased in patients affected by major depressive disorder (MDD) when compared to healthy non-psychiatric controls cohort (p=0.035). Interestingly, pharmacological antidepressant treatments restore normal PE levels. LIMITATIONS: The limitation of the study concerns the relatively small patient samples that could negatively affect the likelihood that a nominally statistically significant finding actually reflects a true effect. CONCLUSIONS: The longitudinal quantification of the serum PE concentration could assist psychiatrists in the diagnosis of antidepressant response efficacy, and the need to modify the therapeutic strategy.

Fluoxetine Protection in Decompression Sickness in Mice is Enhanced by Blocking TREK-1 Potassium Channel with the "spadin" Antidepressant.

In mice, disseminated coagulation, inflammation, and ischemia induce neurological damage that can lead to death. These symptoms result from circulating bubbles generated by a pathogenic decompression. Acute fluoxetine treatment or the presence of the TREK-1 potassium channel increases the survival rate when mice are subjected to an experimental dive/decompression protocol. This is a paradox because fluoxetine is a blocker of TREK-1 channels. First, we studied the effects of an acute dose of fluoxetine (50 mg/kg) in wild-type (WT) and TREK-1 deficient mice (knockout homozygous KO and heterozygous HET). Then, we combined the same fluoxetine treatment with a 5-day treatment protocol with spadin, in order to specifically block TREK-1 activity (KO-like mice). KO and KO-like mice were regarded as antidepressed models. In total, 167 mice (45 WTcont 46 WTflux 30 HETflux and 46 KOflux) constituting the flux-pool and 113 supplementary mice (27 KO-like 24 WTflux2 24 KO-likeflux 21 WTcont2 17 WTno dive) constituting the spad-pool were included in this study. Only 7% of KO-TREK-1 treated with fluoxetine (KOflux) and 4% of mice treated with both spadin and fluoxetine (KO-likeflux) died from decompression sickness (DCS) symptoms. These values are much lower than those of WT control (62%) or KO-like mice (41%). After the decompression protocol, mice showed significant consumption of their circulating platelets and leukocytes. Spadin antidepressed mice were more likely to exhibit DCS. Nevertheless, mice which had both blocked TREK-1 channels and fluoxetine treatment were better protected against DCS. We conclude that the protective effect of such an acute dose of fluoxetine is enhanced when TREK-1 is inhibited. We confirmed that antidepressed models may have worse DCS outcomes, but concomitant fluoxetine treatment not only decreased DCS severity but increased the survival rate.

The peptidic antidepressant spadin interacts with prefrontal 5-HT(4) and mGluR(2) receptors in the control of serotonergic function.

This study investigates the mechanism of action of spadin, a putative fast-acting peptidic antidepressant (AD) and a functional blocker of the K(+) TREK-1 channel, in relation with the medial prefrontal cortex (mPFC)-dorsal raphé (DRN) serotonergic (5-HT) neurons connectivity. Spadin increased 5-HT neuron firing rate by 113%, an augmentation abolished after electrolytic lesion of the mPFC. Among the few receptor subtypes known to modulate TREK-1, the stimulation of 5-HT4 receptors and the blockade of mGluR2/3 ones both activated 5-HT impulse flow, effects also suppressed by mPFC lesion. The combination of spadin with the 5-HT4 agonist RS 67333 paradoxically reduced 5-HT firing, an effect reversed by acutely administering the 5-HT1A agonist flesinoxan. It also had a robust synergetic effect on the expression of Zif268 within the DRN. Together, these results strongly suggest that 5-HT neurons underwent a state of depolarization block, and that the mechanisms underlying the influences exerted by spadin and RS 67333 are additive and independent from each other. In contrast, the mGluR2/3 antagonist LY 341495 occluded the effect of spadin, showing that it likely depends on mPFC TREK-1 channels coupled to mGluR2/3 receptors. These in vivo electrophysiological data were confirmed by in vitro Ca(2+) cell imaging performed in cultured cortical neurons. Altogether, our results indicate that spadin, as a natural compound, constitutes a very good candidate to explore the "glutamatergic path" of fast-acting AD research. In addition, they provide the first evidence of 5-HT depolarization block, showing that the combination of 5-HT activators for strategies of AD augmentation should be performed with extreme caution.

Potentiation of Calcium Influx and Insulin Secretion in Pancreatic Beta Cell by the Specific TREK-1 Blocker Spadin.

Inhibition of the potassium channels TREK-1 by spadin (SPA) is currently thought to be a promising therapeutic target for the treatment of depression. Since these channels are expressed in pancreatic ß-cells, we investigated their role in the control of insulin secretion and glucose homeostasis. In this study, we confirmed the expression of TREK-1 channels in the insulin secreting MIN6-B1 ß-cell line and in mouse islets. We found that their blockade by SPA potentiated insulin secretion induced by potassium chloride dependent membrane depolarization. Inhibition of TREK-1 by SPA induced a decrease of the resting membrane potential (ΔVm ~ 12 mV) and increased the cytosolic calcium concentration. In mice, administration of SPA enhanced the plasma insulin level stimulated by glucose, confirming its secretagogue effect observed in vitro. Taken together, this work identifies SPA as a novel potential pharmacological agent able to control insulin secretion and glucose homeostasis.

Differential phospholipase C-dependent modulation of TASK and TREK two-pore domain K+ channels in rat thalamocortical relay neurons.

KEY POINTS: During the behavioural states of sleep and wakefulness thalamocortical relay neurons fire action potentials in high frequency bursts or tonic sequences, respectively. The modulation of specific K(+) channel types, termed TASK and TREK, allows these neurons to switch between the two modes of activity. In this study we show that the signalling lipids phosphatidylinositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG), which are components of their membrane environment, switch on and shut off TREK and TASK channels, respectively. These channel modulations contribute to a better understanding of the molecular basis of the effects of neurotransmitters such as ACh which are released by the brainstem arousal system. The present report introduces PIP2 and DAG as new elements of signal transduction in the thalamus. The activity of two-pore domain potassium channels (K2P ) regulates the excitability and firing modes of thalamocortical (TC) neurons. In particular, the inhibition of two-pore domain weakly inwardly rectifying K(+) channel (TWIK)-related acid-sensitive K(+) (TASK) channels and TWIK-related K(+) (TREK) channels, as a consequence of the stimulation of muscarinic ACh receptors (MAChRs) which are coupled to phosphoinositide-specific phospholipase C (PLCß), induces a shift from burst to tonic firing. By using a whole cell patch-clamp approach, the contribution of the membrane-bound second messenger molecules phosphatidylinositol 4,5-bisphosphate (PIP2 ) and diacylglycerol (DAG) acting downstream of PLCß was probed. The standing outward current (ISO ) was used to monitor the current through TASK and TREK channels in TC neurons. By exploiting different manoeuvres to change the intracellular PIP2 level in TC neurons, we here show that the scavenging of PIP2 (by neomycin) results in an increased muscarinic effect on ISO whereas increased availability of PIP2 (inclusion to the patch pipette; histone-based carrier) decreased muscarinic signalling. The degree of muscarinic inhibition specifically depends on phosphatidylinositol phosphate (PIP) and PIP2 but no other phospholipids (phosphatidic acid, phosphatidylserine). The use of specific blockers revealed that PIP2 is targeting TREK but not TASK channels. Furthermore, we demonstrate that the inhibition of TASK channels is induced by the application of the DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG). Under current clamp conditions the activation of MAChRs and PLCß as well as the application of OAG resulted in membrane depolarization, while PIP2 application via histone carrier induced a hyperpolarization. These results demonstrate a differential role of PIP2 and DAG in K2P channel modulation in native neurons which allows a fine-tuned inhibition of TREK (via PIP2 depletion) and TASK (via DAG) channels following MAChR stimulation.

Retroinverso analogs of spadin display increased antidepressant effects.

RATIONALE: Although depression is the most common mood disorder, only one third of patients are treated with success. Finding new targets, new drugs, and also new drug intake way are the main challenges in the depression field. Several years ago, we identified a new target with the TWIK-related potassium channel-1 (TREK-1) potassium channel, and more recently, we have discovered a peptide of 17 amino acids with antidepressant properties. This peptide, that we called spadin, can be considered as a new concept in antidepressant drug design. Spadin derives from a larger peptide resulting to a posttranslational maturation of sortilin; consequently, spadin can be considered as a natural molecule. Moreover, spadin acts more rapidly than classical antidepressants and does not induce side effects. OBJECTIVES: In this work, we sought analogs of spadin displaying a better affinity on TREK-1 channels and an increased action duration. METHODS: Analogs were characterized by electrophysiology measurements, by behavioral tests, and by their ability to induce neurogenesis. RESULTS: We identified two retro-inverso peptides that have kept the antidepressant properties of spadin; particularly, they increased the hippocampal neurogenesis after a 4-day treatment. As spadin, these analogs did not induce side effects on either pain, epilepsy processes, or at the cardiac level. CONCLUSIONS: Together, our results indicated that spadin retro-inverso peptides could represent new potent antidepressant drugs. As exemplified by spadin in the field of depression, retro-inverso strategies could represent a useful technique for developing new classes of drugs in a number of pathologies.

Endothelial TWIK-related potassium channel-1 (TREK1) regulates immune-cell trafficking into the CNS.

The blood-brain barrier (BBB) is an integral part of the neurovascular unit (NVU). The NVU is comprised of endothelial cells that are interconnected by tight junctions resting on a parenchymal basement membrane ensheathed by pericytes, smooth muscle cells and a layer of astrocyte end feet. Circulating blood cells, such as leukocytes, complete the NVU. BBB disruption is common in several neurological diseases, but the molecular mechanisms involved remain largely unknown. We analyzed the role of TWIK-related potassium channel-1 (TREK1, encoded by KCNK2) in human and mouse endothelial cells and the BBB. TREK1 was downregulated in endothelial cells by treatment with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Blocking TREK1 increased leukocyte transmigration, whereas TREK1 activation had the opposite effect. We identified altered mitogen-activated protein (MAP) kinase signaling, actin remodeling and upregulation of cellular adhesion molecules as potential mechanisms of increased migration in TREK1-deficient (Kcnk2(-/-)) cells. In Kcnk2(-/-) mice, brain endothelial cells showed an upregulation of the cellular adhesion molecules ICAM1, VCAM1 and PECAM1 and facilitated leukocyte trafficking into the CNS. Following the induction of experimental autoimmune encephalomyelitis (EAE) by immunization with a myelin oligodendrocyte protein (MOG)35-55 peptide, Kcnk2(-/-) mice showed higher EAE severity scores that were accompanied by increased cellular infiltrates in the central nervous system (CNS). The severity of EAE was attenuated in mice given the amyotrophic lateral sclerosis drug riluzole or fed a diet enriched with linseed oil (which contains the TREK-1 activating omega-3 fatty acid α-linolenic acid). These beneficial effects were reduced in Kcnk2(-/-) mice, suggesting TREK-1 activating compounds may be used therapeutically to treat diseases related to BBB dysfunction.

Mapacalcine protects mouse neurons against hypoxia by blocking cell calcium overload.

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment.

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons.

Modulation of the standing outward current (I (SO)) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K(+) (K(2P)) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K(+) (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K(+) (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M(1)AChR-(pirenzepine, MT-7) and M(3)AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCß (PI-PLC) inhibitors (U73122, ET-18-OCH(3)), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5'-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I (SO). Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M(1)AChR/M(3)AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.

A human TREK-1/HEK cell line: a highly efficient screening tool for drug development in neurological diseases.

TREK-1 potassium channels are involved in a number of physiopathological processes such as neuroprotection, pain and depression. Molecules able to open or to block these channels can be clinically important. Having a cell model for screening such molecules is of particular interest. Here, we describe the development of the first available cell line that constituvely expresses the TREK-1 channel. The TREK-1 channel expressed by the h-TREK-1/HEK cell line has conserved all its modulation properties. It is opened by stretch, pH, polyunsaturated fatty acids and by the neuroprotective molecule, riluzole and it is blocked by spadin or fluoxetine. We also demonstrate that the h-TREK-1/HEK cell line is protected against ischemia by using the oxygen-glucose deprivation model.

Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design.

Current antidepressant treatments are inadequate for many individuals, and when they are effective, they require several weeks of administration before a therapeutic effect can be observed. Improving the treatment of depression is challenging. Recently, the two-pore domain potassium channel TREK-1 has been identified as a new target in depression, and its antagonists might become effective antidepressants. In mice, deletion of the TREK-1 gene results in a depression-resistant phenotype that mimics antidepressant treatments. Here, we validate in mice the antidepressant effects of spadin, a secreted peptide derived from the propeptide generated by the maturation of the neurotensin receptor 3 (NTSR3/Sortilin) and acting through TREK-1 inhibition. NTSR3/Sortilin interacted with the TREK-1 channel, as shown by immunoprecipitation of TREK-1 and NTSR3/Sortilin from COS-7 cells and cortical neurons co-expressing both proteins. TREK-1 and NTSR3/Sortilin were colocalized in mouse cortical neurons. Spadin bound specifically to TREK-1 with an affinity of 10 nM. Electrophysiological studies showed that spadin efficiently blocked the TREK-1 activity in COS-7 cells, cultured hippocampal pyramidal neurons, and CA3 hippocampal neurons in brain slices. Spadin also induced in vivo an increase of the 5-HT neuron firing rate in the Dorsal Raphe Nucleus. In five behavioral tests predicting an antidepressant response, spadin-treated mice showed a resistance to depression as found in TREK-1 deficient mice. More importantly, an intravenous 4-d treatment with spadin not only induced a strong antidepressant effect but also enhanced hippocampal phosphorylation of CREB protein and neurogenesis, considered to be key markers of antidepressant action after chronic treatment with selective serotonin reuptake inhibitors. This work also shows the development of a reliable method for dosing the propeptide in serum of mice by using AlphaScreen technology. These findings point out spadin as a putative antidepressant of new generation with a rapid onset of action. Spadin can be regarded as the first natural antidepressant peptide identified. It corresponds to a new concept to address the treatment of depression.