Alaeddine Djillani

Mariel Pietri

Jean Mazella

Catherine Heurteaux

Marc Borsotto
email: borsotto@ipmc.cnrs.fr

FIGHTING AGAINST DEPRESSION WITH TREK-1 BLOCKERS: PAST AND FUTURE. A FOCUS ON SPADIN

Keywords: TREK-1, depression, blockers, spadin, peptide, analogs Brain-derived neurotrophic factor BrdU: 5-Bromo-2´-Deoxyuridine CNS: central nervous system FST: forced swim test GABA: γ-Aminobutyric acid microtubule-associated protein -norepinephrine reuptake inhibitor

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 more than 7 hours. 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.

Introduction

Depression is a devastating mental disorder that affects one in five individuals and is considered as a leading cause of disability worldwide [START_REF] Otte | Major depressive disorder[END_REF]) [START_REF] Nestler | Neurobiology of depression[END_REF]. Thus, the economic burden is huge for governments and particularly for patients suffering from depression [START_REF] Kessler | The costs of depression[END_REF]. Depression is a product of several and complex molecular and cellular mechanisms that are difficult to identify. One of the main common hypotheses that attempt to explain the neurobiology of depression is the imbalance in monoamine levels in the brain known as the "monoaminergic theory of depression" [START_REF] Delgado | Depression: the case for a monoamine deficiency[END_REF]. Presently, the majority of antidepressant (AD) drugs aim to restore the physiological amount of three central monoamines (serotonin (5-HT), norepinephrine (NA), and dopamine (DA)) in the synaptic cleft. Indeed, this is achievable by blocking the serotonin transporter (SERT), norepinephrine transporter (NAT), and dopamine transporter (DAT) responsible for the reuptake of 5-HT, NA, and DA, respectively, in the brain [START_REF] Tatsumi | Pharmacological profile of antidepressants and related compounds at human monoamine transporters[END_REF]. Inhibiting 5-HT and NA degradation through monoamine oxidase (MAO) enzyme increases the synaptic concentrations of monoamines [START_REF] Brandon | Monoamine oxidase inhibitors in depression[END_REF].

However, with their proven efficacy, these AD drugs present a delay of action that remains very long [START_REF] Cipriani | Comparative efficacy and acceptability of 21 antidepressant drugs for the acute treatment of adults with major depressive disorder: a systematic review and network meta-analysis[END_REF] [START_REF] Stassen | Delayed onset of action of antidepressant drugs? Survey of recent results[END_REF]. The late onset of action of current AD drugs takes several weeks to manifest. Given the complexity of the physiopathology of depression, many mechanisms and factors could be responsible for monoaminergic transmission, stress, inflammation, neurogenesis, and neuroplasticity [START_REF] Dean | The neurobiology of depression: An integrated view[END_REF]. Moreover, AD drugs are frequently associated with various adverse effects such as fatigue, anxiety, sexual dysfunction, headache, and nausea. Often, these adverse effects lead patients to discontinue their AD treatment and in some cases can worsen or even cause increased suicide risk [START_REF] Bull | Discontinuing or switching selective serotonin-reuptake inhibitors[END_REF] [START_REF] Sicouri | Sudden cardiac death secondary to antidepressant and antipsychotic drugs[END_REF] [START_REF] Nischal | Suicide and antidepressants: what current evidence indicates[END_REF][START_REF] Sharma | Suicidality and aggression during antidepressant treatment: systematic review and meta-analyses based on clinical study reports[END_REF]. This increased risk could be attributed to either the drug itself, a delayed onset of action, a wrong dosage, or a discontinuity in the treatment [START_REF] Fergusson | Association between suicide attempts and selective serotonin reuptake inhibitors: systematic review of randomised controlled trials[END_REF][START_REF] Otte | Major depressive disorder[END_REF]. To date, the main goals of the new AD strategy in drug design and development are to shorten the latency time for AD activity to manifest and substantially reduce adverse effects [START_REF] Ramaker | Identifying fast-onset antidepressants using rodent models[END_REF]. The most common AD drugs in use currently as a first-line treatment are the selective serotonin reuptake inhibitors (SSRIs) and the serotonin-norepinephrine reuptake inhibitors (SNRIs). Their safety is seriously questioned in a series of studies [START_REF] Cascade | Real-World Data on SSRI Antidepressant Side Effects[END_REF][START_REF] Ferguson | SSRI Antidepressant Medications: Adverse Effects and Tolerability[END_REF][START_REF] Masand | Long-term side effects of newer-generation antidepressants: SSRIS, venlafaxine, nefazodone, bupropion, and mirtazapine[END_REF][START_REF] Montgomery | Tolerability of serotonin norepinephrine reuptake inhibitor antidepressants[END_REF][START_REF] Read | Adverse Effects of Antidepressants Reported by 1,431 people from 38 Countries: Emotional Blunting, Suicidality, and Withdrawal Effects[END_REF]. As a result, the biggest challenge currently is to prescribe efficient AD drugs but with an acceptable tolerance in depressive patients. Newer AD classes have been discovered, such as multimodal AD drugs [START_REF] Richelson | Multi-modality: a new approach for the treatment of major depressive disorder[END_REF]. In addition to SERT inhibition, these drugs also antagonize 5HT3 and 5HT7 receptors and behave as partial agonists of 5HT1B and full agonists of 5HT1A receptors [START_REF] Katona | New generation multi-modal antidepressants: focus on vortioxetine for major depressive disorder[END_REF]. Recently, the fast-acting AD drug ketamine has been identified and might represent a new generation of AD drugs able to counteract depression with a fast onset of action [START_REF] Caraci | New drugs in psychiatry: focus on new pharmacological targets[END_REF][START_REF] Kavalali | How does ketamine elicit a rapid antidepressant response?[END_REF]. However, despite the advanced level of clinical development accomplished by ketamine, this drug licensed as an anesthetic and painkiller has to be handled with extreme caution. A long list of serious adverse effects caused by ketamine was reported in many studies as described in the review of Short et al [START_REF] Short | Side-effects associated with ketamine use in depression: a systematic review[END_REF]. More recently, a study has identified ELK-1 transcription factor, which is an ERK downstream module as a potential target for treating depression. ELK-1 is upregulated in depressive patients.

In mice, selective inhibition of ELK-1 phosphorylation using a 31-amino acid peptide, called TDE, produces AD-like behavior in different tests [START_REF] Apazoglou | Antidepressive effects of targeting ELK-1 signal transduction[END_REF]. To improve AD efficacy, or suppress unwanted adverse effects, new strategies should be considered involving endogenous molecules. This includes essentially peptides that are naturally synthesized in the human body to exert specific actions. Indeed, peptides are involved in numerous biological functions in the cell, mainly as signaling molecules and also as ligands for several types of receptors. Considering their attractive pharmacological properties, peptides constitute an excellent starting point for designing novel therapeutic molecules. Their specificity results in excellent efficacy, safety, and tolerability in humans [START_REF] Fosgerau | Peptide therapeutics: current status and future directions[END_REF].

Growing evidence places therapeutic peptides as a very promising market, and the number of candidate peptides has shown an important increase in clinical trials [START_REF] Uhlig | The emergence of peptides in the pharmaceutical business: From exploration to exploitation[END_REF]. To date, approximately 100 therapeutic peptides are marketed in the United States, Europe, and Japan [START_REF] Kaspar | Future directions for peptide therapeutics development[END_REF]. In a pharmaceutical industry that lacks efficient innovations, peptides constitute a potential alternative in the treatment of numerous diseases and disorders. Furthermore, new targets in depression need to be discovered and validated for innovative molecules. The goals are to improve AD therapy, eliminate adverse effects, and treat patients who are resistant to classical AD drugs. Ion channels represent almost 20% of all the human protein targets [START_REF] Santos | A comprehensive map of molecular drug targets[END_REF]. Until 2015, 177 small molecules and biologic effectors of ion channels had been approved as treatments for several pathologies [START_REF] Santos | A comprehensive map of molecular drug targets[END_REF]. In the present review, we discuss the spadin (or its analogs)-TREK-1 channel interaction that leads to channel blocking, one of the most promising drug-target interactions in term of CNS disorder treatments, mainly in the depression process.

TREK-1 in Depression

TREK-1 channel

The TWIK-related K + channel-1 (TREK-1) is a member of the two-pore domain K + channel family (K2P) [START_REF] Honore | The neuronal background K2P channels: focus on TREK1[END_REF]Lesage & Lazdunski, 1998). As with other K2P channels, TREK-1 channels are responsible for maintaining the neuronal resting membrane potential and controlling action potential duration and also participate in neurotransmitter release [START_REF] Honore | The neuronal background K2P channels: focus on TREK1[END_REF]. TREK-1, TREK-2, and TRAAK are part of lipid and mechanosensitive K2P channel subfamily. TREK-1 and TREK-2 share more than 78% of homology (Lesage & Lazdunski, 2000). In the cell, TREK-1 is regulated by a variety of physical and chemical stimuli [START_REF] Honore | The neuronal background K2P channels: focus on TREK1[END_REF]. Activation of TREK-1 channels can be mediated by membrane stretch [START_REF] Maingret | Mechano-or acid stimulation, two interactive modes of activation of the TREK-1 potassium channel[END_REF][START_REF] Patel | A mammalian two pore domain mechano-gated S-like K+ channel[END_REF], internal acidosis [START_REF] Honore | An intracellular proton sensor commands lipid-and mechano-gating of the K(+) channel TREK-1[END_REF][START_REF] Maingret | Mechano-or acid stimulation, two interactive modes of activation of the TREK-1 potassium channel[END_REF], heat [START_REF] Maingret | TREK-1 is a heat-activated background K( + ) channel[END_REF], lipids [START_REF] Kim | Fatty acid-sensitive two-pore domain K+ channels[END_REF][START_REF] Patel | Properties and modulation of mammalian 2P domain K + channels[END_REF], and polyunsaturated fatty acids such as arachidonic acid (AA) [START_REF] Patel | A mammalian two pore domain mechano-gated S-like K+ channel[END_REF]. Pharmacological opening of the channel is mediated by volatile general anesthetics [START_REF] Patel | Inhalational anesthetics activate two-pore-domain background K+ channels[END_REF] and analgesics such as morphine through the activation of µ-opioid receptors [START_REF] Devilliers | Activation of TREK-1 by morphine results in analgesia without adverse side effects[END_REF]. TREK-1 is downregulated upon stimulation of Gs-and Gq-coupled receptors. In fact, stimulation of 5-HT4 receptors by 5-HT [START_REF] Fink | Cloning, functional expression and brain localization of a novel unconventional outward rectifier K+ channel[END_REF][START_REF] Patel | A mammalian two pore domain mechano-gated S-like K+ channel[END_REF] or the metabotropic glutamate receptors mGluR1 and mGluR5 by glutamate inhibits the TREK-1 channel [START_REF] Chemin | Mechanisms underlying excitatory effects of group I metabotropic glutamate receptors via inhibition of 2P domain K+ channels[END_REF][START_REF] Lopes | PIP2 hydrolysis underlies agonist-induced inhibition and regulates voltage gating of twopore domain K+ channels[END_REF].

TREK-1 is also inhibited by SSRIs such as fluoxetine [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF][START_REF] Kennard | Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine[END_REF] and the endogenous peptide spadin; this is described in detail in this review [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF].

Screening of TREK-1 modulators in vitro

Since its discovery, TREK-1 was shown to play a major role in several physiopathological processes [START_REF] Honore | The neuronal background K2P channels: focus on TREK1[END_REF]. Its widespread presence in a multitude of functions varying from neurologic brain disorders to arrhythmia in the heart confirms the highly attractive target that TREK-1 represents currently. Through the generation of TREK-1 knockout (kcnk2 -/-) mice, it was clearly demonstrated that TREK-1 channels are involved in several pathologies. TREK-1 activation is known to have neuroprotective properties against ischemia or epilepsy [START_REF] Blondeau | Polyunsaturated fatty acids are cerebral vasodilators via the TREK-1 potassium channel[END_REF][START_REF] Heurteaux | TREK-1, a K(+) channel involved in neuroprotection and general anesthesia[END_REF][START_REF] Lauritzen | Polyunsaturated fatty acids are potent neuroprotectors[END_REF]. More interestingly, kcnk2 -/-mice demonstrated that TREK-1 channels are involved in the depression process. Deletion of TREK-1 channels resulted in a depression-resistant phenotype. This particular phenotype was identified by different animal assays for depressive-like behaviors (such as FST, TST, CMST, NSF, or LHT), i.e., an increase in 5-HT neurotransmission and a reduction in elevated corticosterone levels under stress [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]. Together, these data indicate that TREK-1 modulators are of great pharmacological interest. With the aim to efficiently and easily screen TREK-1 modulators, a HEK293 cell line that stably expresses the human TREK-1 channel was generated and named as the hTREK-1/HEK cell line (Moha ou [START_REF] Moha Ou Maati | A human TREK-1/HEK cell line: a highly efficient screening tool for drug development in neurological diseases[END_REF]. Thus, screening TREK-1 activators for characterizing new neuroprotective molecules could be made easier using the hTREK-1/HEK cell line. The hTREK-1/HEK cell line was validated as an efficient pharmacological tool for screening TREK-1 effectors because it responded to various chemical and physical stimuli that modulate TREK-1 activity. In addition, TREK-1 was associated with pain perception [START_REF] Alloui | TREK-1, a K+ channel involved in polymodal pain perception[END_REF]. Consequently, activators of TREK-1 channels displaying in vivo analgesic activity were also screened using the hTREK-1/HEK cell line [START_REF] Vivier | Development of the First Two-Pore Domain Potassium Channel TWIK-Related K+ Channel 1-Selective Agonist Possessing in Vivo Antinociceptive Activity[END_REF]. Another stable TREK-1 transfected HEK cell line was used to study the role of TREK-1 channels in maintaining uterine quiescence during pregnancy [START_REF] Heyman | TREK-1 currents in smooth muscle cells from pregnant human myometrium[END_REF]. Recently, a CHO cell line stably expressing hTREK-1, named as CHO/hTREK-1 cells, was generated to study the effect of overexpression of TREK-1 on cell proliferation [START_REF] Zhang | Over-expressed human TREK-1 inhibits CHO cell proliferation via inhibiting PKA and p38 MAPK pathways and subsequently inducing G1 arrest[END_REF]. The design of the hTREK-1/HEK cell line was also of great importance for research and screening of new and specific TREK-1 inhibitors with AD properties [START_REF] Borsotto | Targeting two-pore domain K(+) channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept[END_REF][START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF][START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF][START_REF] Veyssiere | Retroinverso analogs of spadin display increased antidepressant effects[END_REF].

Small molecule inhibitors of TREK-1

Selective Serotonin Reuptake Inhibitors

The monoaminergic hypothesis of depression has driven a number of research laboratories to search for new molecules that block 5-HT reuptake. Fluoxetine was approved as an AD in 1986, and clinical trials showed a clear improvement in adverse effects previously observed with tricyclics [START_REF] Perez-Caballero | Fluoxetine: a case history of its discovery and preclinical development[END_REF]. The AD activity was attributed to the high-affinity blockade of SERT, thereby leading to an increase in the amount of 5-HT in the synapses [START_REF] Wong | A selective inhibitor of serotonin uptake: Lilly 110140, 3-(p-trifluoromethylphenoxy)-Nmethyl-3-phenylpropylamine[END_REF].

At clinical concentrations, TREK-1 channels were significantly blocked by fluoxetine and its active metabolite norfluoxetine in a concentration-dependent manner, and the IC50s were 19 µM and 9 µM, respectively [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF][START_REF] Kennard | Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine[END_REF] (Table 1). TREK-1 inhibition seems to be state dependent because norfluoxetine binds to the fenestration in TREK-1 that is only available in the down state (when the lower sections of M2, M3, and M4 transmembrane domains project into the cytoplasm) corresponding to the channel lower activity [START_REF] Dong | K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac[END_REF].

However, when TREK-1 is activated by stretch or AA, the channel conformation changes to the up state (when lower sections of M2, M3, and M4 transmembrane domains project into the membrane), which suppresses the fenestration and does not allow norfluoxetine to bind [START_REF] Dong | K2P channel gating mechanisms revealed by structures of TREK-2 and a complex with Prozac[END_REF][START_REF] Kennard | Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine[END_REF]. In a recent study, other SSRIs have been shown to be potent blockers of TREK-1 and TREK-2 channels in HEK-293 cells and HT-22 neuronal cells. In addition to fluoxetine, TREK-1 inhibition was also observed with paroxetine and citalopram, two other commonly prescribed AD drugs [START_REF] Kim | Activation of TREK-1, but Not TREK-2, Channel by Mood Stabilizers[END_REF] (Table 1).

Other inhibitors of TREK-1 channels

Few molecules were identified in the literature as potent TREK-1 channel blockers (Table 1). SID1900, a small molecule screened from a large library of 487 compounds, has been shown to block TREK-1 channels in a manner similar to that of spadin and to induce an AD-like behavior in a rat model of chronic unpredictable mild stress [START_REF] Ye | TREK1 channel blockade induces an antidepressant-like response synergizing with 5-HT1A receptor signaling[END_REF]. SID1900 inhibits TREK-1 with an IC50 of 29.72 µM, which largely exceeds that obtained with spadin (peptide #1 Table 2) (IC50=40 nM) [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]. Furthermore, antipsychotic drugs have been shown to be modulators of TREK-1 channels [START_REF] Thummler | Antipsychotics inhibit TREK but not TRAAK channels[END_REF]. Typical and atypical antipsychotics such as fluphenazine, chlorpromazine, haloperidol, loxapine, and clozapine inhibit TREK-1 and TREK-2 channels in a dose-dependent manner without affecting TRAAK channels [START_REF] Kim | Activation of TREK-1, but Not TREK-2, Channel by Mood Stabilizers[END_REF][START_REF] Thummler | Antipsychotics inhibit TREK but not TRAAK channels[END_REF] (Table 1). In contrast to antipsychotic drugs, mood stabilizers such as lithium chloride, valproate, gabapentin, and carbamazepine activate TREK-1 channels but have no effect on TREK-2 channels [START_REF] Kim | Activation of TREK-1, but Not TREK-2, Channel by Mood Stabilizers[END_REF]. Dihydropyridine Ca 2+ channel antagonists such as amlodipine and niguldipine are nonspecific blockers of TREK-1 channels (Table 1).

They inhibit TREK-1 channels with an IC50 of 0.43 and 0.75 µM, respectively [START_REF] Liu | Potent inhibition of native TREK-1 K+ channels by selected dihydropyridine Ca2+ channel antagonists[END_REF]. In vitro, they potently block calcium-induced vascular smooth muscle contractions with IC50 of 1.9 and 4.1 nM for amlodipine and nifedipine, respectively [START_REF] Burges | Calcium channel blocking properties of amlodipine in vascular smooth muscle and cardiac muscle in vitro: evidence for voltage modulation of vascular dihydropyridine receptors[END_REF]. In addition, L-methionine was reported to decrease the probability of TREK-1 opening in a cell-attached patch-clamp configuration [START_REF] Baker | Methionine and its derivatives increase bladder excitability by inhibiting stretchdependent K(+) channels[END_REF][START_REF] Lei | Response of the human detrusor to stretch is regulated by TREK-1, a two-pore-domain (K2P) mechano-gated potassium channel[END_REF] (Table 1). This amino acid was used as a tool to investigate the role of TREK-1 in uterine contraction [START_REF] Yin | Progesterone inhibits contraction and increases TREK-1 potassium channel expression in late pregnant rat uterus[END_REF] and in controlling bladder smooth muscle cell excitability following contraction [START_REF] Baker | Methionine and its derivatives increase bladder excitability by inhibiting stretchdependent K(+) channels[END_REF]. However, L-methionine inhibition on TREK-1 has been shown to have contrast in another study where no TREK-1 inhibitory effect was observed in rat colon smooth muscle cells after activation with AA [START_REF] Gil | Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon[END_REF]. The neuroprotective compound 3-n-Butylphthalide (NBP) and its racemic form dl-NBP were reported to block TREK-1 [START_REF] Ji | Novel neuroprotectant chiral 3-n-butylphthalide inhibits tandem-pore-domain potassium channel TREK-1[END_REF]. Recently, another NBP analog, named as lig4-4, was described as a specific blocker of TREK-1 [START_REF] Wang | Lig4-4 selectively inhibits TREK-1 and plays potent neuroprotective roles in vitro and in rat MCAO model[END_REF]. The authors presumed that the neuroprotective effects of lig4-4 observed in ischemic stroke might be related to TREK-1 inhibition [START_REF] Wang | Lig4-4 selectively inhibits TREK-1 and plays potent neuroprotective roles in vitro and in rat MCAO model[END_REF]. This hypothesis shows total contradiction to data published more than 10 years ago that showed that the opening of TREK-1 is neuroprotective (Heurteaux et al., 2006a). However, neither NBP nor lig4-4 can be considered as specific blockers for TREK-1 because lig4-4 also inhibits hERG, Kv1.5, Kv2.1, Kv3.1, and neuronal Na + and Ca 2+ channels [START_REF] Wang | Lig4-4 selectively inhibits TREK-1 and plays potent neuroprotective roles in vitro and in rat MCAO model[END_REF].

Birth of an idea…

Approximately 10 years ago, TREK-1 was described as a new target in depression [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]. This K2P mechanosensitive channel is mainly expressed in the prefrontal cortex and the hippocampus [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF][START_REF] Medhurst | Distribution analysis of human two pore domain potassium channels in tissues of the central nervous system and periphery[END_REF]. These regions are known to mediate cognitive aspects of depression, such as memory impairment, feeling of worthlessness, guilt, and suicidality [START_REF] Nestler | Neurobiology of depression[END_REF][START_REF] Otte | Major depressive disorder[END_REF]. TREK-1 is also expressed in the amygdala, hypothalamus, and in the striatum, particularly in the nucleus accumbens to mediate memory of emotional events. Finally, TREK-1 is abundant in GABA (γ-aminobutyric acid)containing neurons of the caudate nucleus and putamen [START_REF] Hervieu | Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS[END_REF] and in hippocampal glutamatergic neurons [START_REF] Medhurst | Distribution analysis of human two pore domain potassium channels in tissues of the central nervous system and periphery[END_REF]. Investigation of the role of TREK-1 channel in the physiopathology of depression by using the knockout of Kcnk2 (the gene coding for TREK-1 channel) in mice demonstrated a depressionresistant phenotype [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF] in five assays for depression-like behaviors [START_REF] Cryan | The ascent of mouse: advances in modelling human depression and anxiety[END_REF]: Tail Suspension Test (TST), Forced Swim Test (FST), Conditioned Suppression of Motility Test (CSMT), Learned Helplessness Test (LHT), and Novelty-Suppressed Feeding Test (NSF). In TREK-1-deficient mice, neurogenesis induced by the well-known SSRI fluoxetine was significantly increased compared to the wild-type mice [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]. However, the proliferation of newborn cells manifested only after 21 days of treatment.

On the other hand, deletion of the Kcnk2 gene enhanced the firing of 5-HT neurons in the dorsal raphe nucleus [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]. Knowing the importance of 5-HT in the neurobiology of depression "Monoaminergic Theory of Depression" [START_REF] Duman | A molecular and cellular theory of depression[END_REF] and because TREK-1 is inhibited by SSRIs such as fluoxetine, this K2P channel has been considered as a serious candidate to play a key role in the physiopathology of depression [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF].

In humans, Star*D study has identified an association between the existence of four genetic variants (single nucleotide polymorphisms [SNPs]) in the TREK-1 locus and resistance to multiple AD classes [START_REF] Perlis | Pharmacogenetic Analysis of Genes Implicated in Rodent Models of Antidepressant Response: Association of TREK1 and Treatment Resistance in the STAR(*)D Study[END_REF]. Another study showed that an SNP at the 3'-untranslated region on exon 7 of the kcnk2 gene could be associated with both depression incidence and poor treatment efficacy [START_REF] Liou | Support for the involvement of the KCNK2 gene in major depressive disorder and response to antidepressant treatment[END_REF] .

Although no brain imaging studies in depressive patients have been published yet, a study suggested that some TREK-1 genotypes in humans can be associated with a depression-resistant phenotype [START_REF] Dillon | Variation in TREK1 gene linked to depression-resistant phenotype is associated with potentiated neural responses to rewards in humans[END_REF]. Taken together, these studies in humans strengthen the idea that TREK-1 represents a crucial target in the field of depression and the search for selective blockers of TREK-1 might potentially lead to a new generation of AD drugs.

SORTing TREK-1...

Within the neuron, the TREK-1 channel forms a complex made of the A-kinaseanchoring protein AKAP150 [START_REF] Sandoz | AKAP150, a switch to convert mechano-, pHand arachidonic acid-sensitive TREK K(+) channels into open leak channels[END_REF] and the microtubule-associated protein Mtap2 [START_REF] Sandoz | Mtap2 is a constituent of the protein network that regulates twik-related K+ channel expression and trafficking[END_REF]. Both proteins regulate the sorting of TREK-1 to the plasma membrane. However, these partner proteins are not unique. In 2010, another crucial interacting protein, named as sortilin, was discovered. Sortilin regulates, transports, and targets the TREK-1 channel to the plasma membrane where it exerts its role as a background potassium channel [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF].

Two decades ago, sortilin, a 95 kDa protein, was identified as a sorting molecule in the human brain [START_REF] Petersen | Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography[END_REF]. It was also described shortly later in another study as the neurotensin receptor-3 (NTSR-3) [START_REF] Mazella | The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor[END_REF]. Sortilin/NTSR-3 binds a number of ligands such as neurotensin (NT), precursor of the nerve growth factor (proNGF) [START_REF] Nykjaer | Sortilin is essential for proNGF-induced neuronal cell death[END_REF], lipoprotein lipase [START_REF] Nielsen | Sortilin/neurotensin receptor-3 binds and mediates degradation of lipoprotein lipase[END_REF], and propeptide (PE) (Munck Petersen et al., 1999). In the Golgi network, the posttranslational cleavage of prosortilin (precursor of sortilin) by the protein convertase furin results in mature sortilin and the release of a 44-amino acid peptide named as PE (Fig. 1) [START_REF] Munck Petersen | Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding[END_REF]. Sortilin/NTSR-3 co-localizes with TREK-1 in numerous brain areas involved in mood, such as the prefrontal cortex, hippocampus, striatum, amygdala, and hypothalamus [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. In addition, physical interaction between TREK-1 and sortilin/NTSR-3 was characterized by pull down experiments. It has been showed that TREK-1 expression at the plasma membrane is greatly enhanced in the presence of sortilin/NTSR-3 [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. Although present in low amounts at the plasma membrane in the absence of sortilin, TREK-1 channels can be modulated by the different effectors described above.

Furthermore, an interesting observation from our team showed that sortilin/NTSR-3deficient mice represent a depression-resistant phenotype similar to the behavior of kcnk2 -/-mice [START_REF] Moreno | Altered Trek-1 Function in Sortilin Deficient Mice Results in Decreased Depressive-Like Behavior[END_REF]. Arguments cited above support the involvement of both TREK-1 and sortilin/NTSR-3 in the physiopathology of depression.

Discovery of SPADIN

Sortilin-derived Peptide with Antidepressant properties

PE is a 44-amino acid peptide that binds with high affinity (Kd ~20-30 nM) to the mature sortilin/NTSR-3 (Munck Petersen et al., 1999). The peptide sequence required for the binding of PE to sortilin/NTSR-3 was identified as Gln 1 -Arg 28 (Fig. 1).

Moreover, the PE fragment corresponding to Gln 1 -Arg 16 had lower affinity in binding the mature sortilin [START_REF] Westergaard | Functional organization of the sortilin Vps10p domain[END_REF]. In this study, a 17-amino acidcontaining peptide was designed and named as spadin, acronym generated from Sortilin-derived Peptide with Antidepressant properties. Spadin contains the main fragment of PE, Trp 17 -Arg 28 , capable of binding to sortilin/NTSR-3 and stabilized by the sequence Ala 12 -Pro 13 -Leu 14 -Pro 15 -Arg 16 added upstream to finally generate a 17amino acid peptide corresponding to the sequence Ala 12 -Arg 28 or PE 12-28 (Fig. 1, Table 2, peptide #1) [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. PE has been shown to antagonize the effects of NT on cell migration of the human microglia cell line C13NJ that expresses the NTSR-3 subtype, exclusively [START_REF] Martin | Involvement of the neurotensin receptor-3 in the neurotensin-induced migration of human microglia[END_REF]. Similarly to PE, spadin (PE 12-28) is able to bind with an identical affinity (Kd=8 nM) to sortilin/NTSR-3 [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. The similarity between PE and spadin to bind to sortilin/NTSR-3 is extended to their functional properties. For example, spadin totally blocks cell migration by displacing NT from the binding site of sortilin/NTSR3 [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF].

Given the role of TREK-1 channels in depression [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF], the question that arose was to determine whether PE or spadin could regulate TREK-1 channels and consequently generate a depression-resistant phenotype in mice. To answer this question, spadin was tested on TREK-1-expressing COS-7 cells in electrophysiological experiments. In the patch-clamp technique using whole-cell configuration, TREK-1 was first activated by 10 µM of AA, and then, when spadin was applied extracellularly, TREK-1 was potently blocked by spadin with an IC50 of 70.7 nM (Tables 1 and2) [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. TREK-1 blockade was confirmed on other type of cell lines such as the hTREK-1/HEK (IC50=40 nM) or the pancreatic β-TC3 cells [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF][START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. The direct blocking of TREK-1 channel activity was demonstrated on the h-TREK-1/HEK cell line by using excised patches in an inside-out configuration [START_REF] Gil | Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon[END_REF]. On brain slices, endogenous TREK-1 currents recorded in the hippocampal CA3 pyramidal cells were also activated by AA and strongly blocked by spadin [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. More interestingly, on hippocampal brain slices, the spadin effect totally disappeared in TREK-1-deficient mice, which confirms TREK-1 as a target for spadin [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF].

TREK-1 is known to be highly expressed in the dorsal raphe nucleus (DRN) [START_REF] Medhurst | Distribution analysis of human two pore domain potassium channels in tissues of the central nervous system and periphery[END_REF]) and kcnk2 -/-mice showed an enhanced serotonergic neurotransmission [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]. To address the question whether pharmacological blockade of TREK-1 by spadin affects 5-HT neurotransmission, mice were given a 10 µM dose of spadin as an i.p injection and the unitary extracellular activity of serotonergic neurons was recorded in anesthetized animals.

Data showed that spadin potentiates 5-HT neurotransmission firing in vivo [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF], similar to the activity of the 5-HT neurons in kcnk2 -/-mice [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]. To investigate further, we addressed the question whether blocking TREK-1 and increasing 5-HT firing in the DRN by spadin could have an impact on mouse behavior. Spadin was administered to mice in acute and subchronic treatments and AD activity was assessed using several mouse models of depression.

Spadin displays AD properties in mice in depression-like behavior tests (FST, TST, CSMT, LHT, and NSF). Moreover, spadin treatments generate two canonical effects of AD drugs: the increase in 5-HT neurotransmission and the induction of neurogenesis [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. These results confirmed the previous data obtained with Kcnk2 -/-mice, whose main phenotype is resistance to depression [START_REF] Heurteaux | Deletion of the background potassium channel TREK-1 results in a depression-resistant phenotype[END_REF]. In addition, subchronic treatment with spadin results in increasing CREB activation and hippocampal neurogenesis. This effect is outstanding, as it occurs only after a 4 day treatment in contrast to SSRIs that need 3-4 weeks of treatment to increase hippocampal neurogenesis [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF].

Moreover, in vitro (in cortical neurons) and in vivo (in the hippocampus), studies showed that spadin increases both mRNA and protein expression of markers of synaptogenesis, such as the post-synaptic density protein-95 (PSD-95) or synapsin [START_REF] Devader | In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin[END_REF]. These data indicated that spadin increases the number of functional neurons. This observation was supported by the fact that spadin treatment significantly increases the number of mature spines on axons [START_REF] Devader | In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin[END_REF].

Taken together, these data confirmed that the newly generated neurons by spadin are indeed functional and able to interact within the neuronal complex network.

Spadin is a specific and safe blocker of TREK-1

Despite the high homology in structure, function, and regulation between TREK-1, TREK-2, and TRAAK channels (Lesage & Lazdunski, 2000), spadin specifically blocks TREK-1, as no effect is observed on the other stretch-activated K2P channels such as TREK-2 and TRAAK. TASK-1 and TRESK, two other members of the K2P channel family, are also insensitive to spadin (Table 1) [START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF].

As TASK-1 is involved in inflammation [START_REF] Bittner | TASK1 modulates inflammation and neurodegeneration in autoimmune inflammation of the central nervous system[END_REF] and apoptosis [START_REF] Lauritzen | K+-dependent cerebellar granule neuron apoptosis. Role of task leak K+ channels[END_REF][START_REF] Leithner | TASK-1 Regulates Apoptosis and Proliferation in a Subset of Non-Small Cell Lung Cancers[END_REF] and TRESK in migraine [START_REF] Lafreniere | Migraine: Role of the TRESK two-pore potassium channel[END_REF] and pain [START_REF] Marsh | Leak K(+) channel mRNAs in dorsal root ganglia: relation to inflammation and spontaneous pain behaviour[END_REF][START_REF] Tulleuda | TRESK channel contribution to nociceptive sensory neurons excitability: modulation by nerve injury[END_REF], the absence of spadin effect on these channels might be beneficial.

TREK-1 was also shown to play an important role in pain perception [START_REF] Alloui | TREK-1, a K+ channel involved in polymodal pain perception[END_REF]. Kcnk2 -/-mice are more sensitive to thermal pain [START_REF] Alloui | TREK-1, a K+ channel involved in polymodal pain perception[END_REF]. Here again, spadin is unable to modify pain perception in tail flick and hot plate tests [START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF].

Undoubtedly, TREK-1 plays a crucial role in the regulation of neuronal excitability [START_REF] Heurteaux | TREK-1, a K(+) channel involved in neuroprotection and general anesthesia[END_REF]. TREK-1-mediated neuroprotection against epilepsy and cerebrovascular diseases is not affected by spadin treatment. Spadin does not increase pentylenetetrazol (PTZ) or kainate-induced seizures [START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF]. Furthermore, a 3-week spadin treatment does not increase the infarct size after focal ischemia [START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF].

Finally, at the cardiovascular level, long-term treatment by spadin has no effect on systolic blood pressure and heart pulses [START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF].

One of the major challenges in the development of safe drugs is the early detection of prolongation of the QT interval, which causes Torsades de pointes (TdP). Long QT-inducing drugs block two types of potassium currents: the rapid (IKr) and the slow (IKs) potassium currents [START_REF] Cheng | Two components of delayed rectifier K+ current in heart: molecular basis, functional diversity, and contribution to repolarization[END_REF][START_REF] Sanguinetti | Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents[END_REF]. IKr current is carried by the hERG channel. However, IKs activity requires the association of KCNQ1 with KCNE1 [START_REF] Barhanin | K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current[END_REF][START_REF] Sanguinetti | Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel[END_REF]. As IKr and IKs are essential components for normal cardiac function, drug-acquired QT interval prolongation due to hERG inhibition causes an increase in sudden death [START_REF] Brown | Drugs, hERG and sudden death[END_REF]. Consequently, several cardiovascular and non-cardiovascular drugs have been withdrawn from the market [START_REF] Finlayson | Acquired QT interval prolongation and HERG: implications for drug discovery and development[END_REF]. Presently, all drug candidates should be tested in vitro to check whether or not they inhibit hERG channels [START_REF] Chen | Cardiac Delayed Rectifier Potassium Channels in Health and Disease[END_REF]. With the aim to develop spadin as an approved drug in the treatment of depression, this peptide was tested for a possible hERG channel inhibition. Spadin does not affect hERG channel biophysical properties, as the use of 10 µM or higher concentrations of spadin do not show any modifications of IKr or IKs [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF][START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF]. Taken together, these observations confirm spadin as a safe molecule for further use in clinic. The potent AD properties of spadin have given rise to the patent published under the no. US8252748B2 [START_REF] Mazella | Peptide derived from neurotensin receptor 3 and use thereof in the treatment of psychiatric diseases[END_REF].

Increasing spadin efficacy and in vivo bioavailability

In FST, spadin AD activity lasts for 7 h after a single i.p injection [START_REF] Veyssiere | Retroinverso analogs of spadin display increased antidepressant effects[END_REF]. To improve in vivo stability of spadin and prolong AD activity beyond 7 h after an acute treatment, different strategies have been thoroughly considered, such as the retro-inverso (RI) strategy, peptide cyclization, amino acid replacement, protection of C-and N-terminal ends of the peptides, and finally shortening of the spadin sequence. The RI strategy consists of changing the amino acid configuration from L to D. At the same time, the amino acid sequences are inverted. RI technology was shown in many studies to increase the resistance of peptides to proteolysis and thus improve their bioavailability in the blood [START_REF] Chorev | Recent developments in retro peptides and proteins--an ongoing topochemical exploration[END_REF][START_REF] Chorev | Partially modified retro-inverso-enkephalinamides: topochemical long-acting analogs in vitro and in vivo[END_REF]. Eleven spadin analogs including RI spadin analogs were synthesized and screened on the hTREK-1/HEK cell line by the patch-clamp technique (Table 2, peptides #2 to #12) Two RI analogs were identified: analog 3 (Ac-RI-PE 12-28, peptide # 3) and 8 (Ac-RI-PE 1-28, peptide # 8) (Fig. 1,Table 2). They display a better inhibition potency for TREK-1 channel activity, IC50 were 11.5 ± 0.59 nM and 9.95 ± 0.85 nM for analog 3 and analog 8, respectively, compared to 56.39 ± 0.01 nM for spadin [START_REF] Veyssiere | Retroinverso analogs of spadin display increased antidepressant effects[END_REF].

The analogs 3 and 8 share the same AD properties with spadin after acute or subchronic treatments. Similar to spadin, analogs 3 and 8 significantly reduce the immobility time in the FST [START_REF] Veyssiere | Retroinverso analogs of spadin display increased antidepressant effects[END_REF]. Moreover, in the NSF, the two RI analogs shorten the latency time for eating in mice. More interestingly, these analogs induce hippocampal neurogenesis.

One of the challenges in terms of development is to improve the in vivo stability of spadin. RI analogs prolong the AD activity by three times compared to spadin [START_REF] Veyssiere | Retroinverso analogs of spadin display increased antidepressant effects[END_REF]. Specifically, analog 3 does not produce any adverse effects on pain, epilepsy, or arrhythmia. RI analogs including analog 3 and analog 8 were patented for the treatment of depression under the number WO2015110915A2 [START_REF] Gaudriault | Retro-inverso analogs of spadin display increased antidepressant effects[END_REF].

Other analogs were designed by the RI strategy, such as analog 13 (G/A-PE 12-28, Table 2 peptide #13), analog 14 (RI-G/A-PE 12-28, Table 2 peptide #14),analog 15 (c(RI-PE 12-28), Table 2 peptide #15), and analog 16 (a tandem of c(RI-PE 12-28), Table 2 peptide #16). The peptide sequences are depicted in Figure 1.

Analog 13 contains the same sequence as spadin, but the three glycine residues were replaced by three alanine residues. Analog 14 is the RI of analog 13. Both analogs 13 and 14 are able to strongly inhibit TREK-1 channels (83.50% ± 9.76%, n=8, p=0.94), (72.15% ± 11.75%, n=9, p=0.36), respectively, compared to spadin (87.08% ± 7.32%, n=8) (Fig. 2 a,b). More interestingly, both analogs conserve their AD activity in FST when injected intravenously (i.v.). The immobility time is significantly decreased (120.4 ± 9.7 s, n=10, p=0.0049 and 127.7 ± 8.32 s, n=10, p=0.0105, respectively) for analogs 13 and 14 compared to saline (160.6 ± 7.97 s, n=10) (Fig. 3a).

Peptide cyclization is one of the common strategies used to increase peptide stability and efficacy [START_REF] Adessi | Converting a peptide into a drug: strategies to improve stability and bioavailability[END_REF]. It has been shown that cyclization could increase peptide resistance against proteolytic degradation that subsequently enhances the peptide bioavailability [START_REF] Wang | Rational design and synthesis of an orally bioavailable peptide guided by NMR amide temperature coefficients[END_REF]. Therefore, the strategy consisted of cyclizing analog 3 alone (analog 15, Table 2 peptide #15) or in tandem (analog 16, Table 2 peptide #16) sequence (Fig. 1) to study the impact of the cyclization on peptides crossing the blood-brain barrier (BBB). Using the whole-cell configuration of the patch-clamp technique, both analogs 15 and 16 inhibit TREK-1 current with different potencies (67.24% ± 3.41%, n=9, p=0.046 and 91.25% ± 8.14%, n=10, p=0.67), respectively (Fig. 2). However, in the FST, when both analogs are administered through the i.v. route, neither analog 15 nor analog 16 is able to produce a significant decrease in immobility time (157 ± 8.09 s, n=10, p=0.755 and 146.7 ± 12.99 s, n=10, p=0.374, respectively) compared to saline (160.6 ± 7.97 s, n=10) (Fig. 3a). It appears that the lack of activity of analog 15 is due to its inability to cross the BBB, as it displays AD activity only when injected directly in the brain by an intracerebroventricular route (i.c.v.) (Fig. 3b).

Shortened spadin analogs with antidepressant activity

Spadin blood degradation products revealed by high-pressure liquid chromatography (HPLC) consist of at least two short peptides PE 12-27 (Table 2, peptide #17) and PE 14-25 (Table 2, peptide #18) [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]. On the basis of this analysis, several short analogs were designed and screened on the hTREK-1/HEK cell line.

PE 22-28 (Table 2, peptide #21) is the shortest, most efficient sequence capable of blocking the TREK-1 channel with higher potency (IC50=0.12 nM versus IC50=40 nM for spadin (PE 12-28)) (Fig. 1). Then, PE 22-28 (peptide #21) was used as the core peptide to design other analogs with different N-and C-terminal end modifications.

These PE 22-28 analogs abolish or maintain TREK-1 inhibition on the basis of the nature of the chemical group attached to C or N-terminus (Table 2) [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF].

With PE 22-28, two other analogs were retained for further studies, as they inhibit the TREK-1 channel with a higher potency. They correspond to the PE 22-28, where Gly 22 is replaced by an Ala 22 (G/A-PE 22-28, Table 2 peptide #27) and its biotinylated derivative (biotin-G/A-PE 22-28, Table 2 peptide #28) (Fig. 1). Their TREK-1inhibiting potencies are IC50=0.10 nM and IC50=1.2 nM for G/A-PE 22-28 and biotin-G/A-PE 22-28, respectively [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]. This represents, respectively, a 400-and 33-fold increase in TREK-1 inhibition potency in comparison to spadin.

Current blockade is specific to TREK-1, as TREK-2, TRAAK, TRESK, and TASK-1 channels are not inhibited by these shortened peptides. How about their ability to counteract depression behavior? Does shortening spadin sequence and replacing Gly 22 by Ala 22 have any consequences on mice behavior? To answer these questions, a series of behavioral studies on mice treated with short spadin analogs were performed [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF].

These candidate peptides display AD properties after acute and 4 day subchronic treatments in FST, NSF, and LHT. More interestingly, similar to spadin, they produce an AD-like behavior regardless of the route of administration. They efficiently reduce the immobility time in the FST after i.p, i.v., and gavage administration [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]. Similar to spadin, hippocampal neurogenesis is increased as revealed by BrdU (5-Bromo-2´-Deoxyuridine) labeling with a prominent effect of G/A-PE 22-28 (Table 2, peptide #27). At the same time, neurogenesis induced by spadin short analogs is consistent with the increase in PSD-95 expression, a marker of synaptogenesis [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF].

One of the most important effects observed with short spadin analogs is the improvement in terms of in vivo stability compared to spadin. Indeed, G/A-PE 22-28 (21h) and biotin-G/A-PE 22-28 (23h) prolong significantly the action duration of spadin (only 7h) as revealed by FST [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]. This is extremely promising and interesting, as the ultimate goal in the near future is to test them in clinical trials and eventually commercialize these long-lasting peptides as ADs of a new generation (Fig. 4).

However, for the clinical development of any molecule, whether a small drug or a therapeutic peptide, it should be screened to detect any change in the QT interval. PE 22-28 and analogs do not modify hERG channel activity, which render them more specific and safer for a possible further pharmaceutical development [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF].

Spadin and analogs as biomarkers of Major Depressive Disorder

To clinically predict patients with major depressive disorder (MDD) who respond to classical AD treatment and routinely monitor AD activity, there is a pressing need to validate biomarkers for MDD. This will allow an inexpensive diagnosis and predictive test for an MDD response to facilitate patient follow-up [START_REF] Woods | Biomarkers in major depressive disorder: the role of mass spectrometry[END_REF]. Thus far, there has been no validated biomarker for MDD, and clinical diagnosis is only symptomatic. Recently, various candidates as biomarkers for MDD were described in the literature, such as inflammatory mediators, growth factors, neurotransmitters, neurotrophic factors, and metabolic biomarkers [START_REF] Gururajan | Molecular biomarkers of depression[END_REF][START_REF] Strawbridge | Biomarkers for depression: recent insights, current challenges and future prospects[END_REF]. Many preclinical studies have pointed out the crucial role of BDNF in the physiopathology of MDD, as BDNF serum levels are lower in patients with MDD than in healthy volunteers or MDD-medicated patients [START_REF] Aydemir | Brain-derived neurotrophic factor (BDNF) changes in the serum of depressed women[END_REF][START_REF] Aydemir | Serum brainderived neurotrophic factor level in dysthymia: a comparative study with major depressive disorder[END_REF][START_REF] Gervasoni | Partial normalization of serum brain-derived neurotrophic factor in remitted patients after a major depressive episode[END_REF][START_REF] Gonul | Effect of treatment on serum brain-derived neurotrophic factor levels in depressed patients[END_REF][START_REF] Huang | Serum brain-derived neurotrophic factor levels in patients with major depression: effects of antidepressants[END_REF]Karege et al., 2005;[START_REF] Monteleone | Decreased levels of serum brain-derived neurotrophic factor in both depressed and euthymic patients with unipolar depression and in euthymic patients with bipolar I and II disorders[END_REF][START_REF] Piccinni | Plasma and serum brain-derived neurotrophic factor (BDNF) in depressed patients during 1 year of antidepressant treatments[END_REF][START_REF] Shimizu | Alterations of serum levels of brain-derived neurotrophic factor (BDNF) in depressed patients with or without antidepressants[END_REF][START_REF] Yoshimura | Effects of paroxetine or milnacipran on serum brain-derived neurotrophic factor in depressed patients[END_REF].

It was already demonstrated that sortilin is an important regulator of BDNF sorting and trafficking to the secretory pathways [START_REF] Chen | Sortilin controls intracellular sorting of brainderived neurotrophic factor to the regulated secretory pathway[END_REF]. A clinical study confirmed the correlation between circulating levels of sortilin, BDNF, and MDD [START_REF] Buttenschon | Increased serum levels of sortilin are associated with depression and correlated with BDNF and VEGF[END_REF]. A recent study was conducted with a cohort of 37 patients suffering from MDD and treated with an AD drug for 12 weeks. They were compared to 49 healthy volunteers [START_REF] Devader | Serum sortilin-derived propeptides concentrations are decreased in major depressive disorder patients[END_REF]. Using the dosing alpha-screen method validated for PE detection in the serum [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF], the concentrations of PE, spadin (PE 12-28, Table 2 peptide #1), PE 12-27 (Table 2 peptide #17), and PE 14-25 (Table 2 peptide #18) were measured with the aim to find a correlation between the serum levels of PE peptides in healthy and patients with MDD whether or not treated with AD drugs. Serum concentrations of the PE peptides are significantly decreased in patients with MDD. Interestingly, PE peptide levels are partially but significantly restored after a 12 week treatment with an AD drug, thus indicating a correlation between the serum levels of PE-like activity and the mood of patients.

Recently, Buttenschon et al. conducted a new study, and they concluded that sortilin could not serve as a biomarker to follow AD treatment in patients with MDD [START_REF] Buttenschon | Investigation of serum levels of sortilin in response to antidepressant treatment[END_REF]. At this stage, it is important to underline that the release of soluble sortilin (as measured in the Buttenschon's work) depends on matrix metalloproteinases (MMPs) [START_REF] Navarro | Shedding of the luminal domain of the neurotensin receptor-3/sortilin in the HT29 cell line[END_REF], whereas PE release depends on the intracellular maturation of sortilin by furin [START_REF] Devader | Serum sortilin-derived propeptides concentrations are decreased in major depressive disorder patients[END_REF]. To validate whether or not PE and sortilin could be used as biomarkers in MDD, further complete clinical studies, which include larger cohorts of patients with MDD whether or not treated with AD drugs, need to be performed in the future. Moreover, advances in developing specific antibodies able to recognize the short peptide sequences with higher sensitivity could guide research toward new biomarkers of MDD.

Conclusions and perspectives

The main advantages that peptides present compared to xenobiotics are their high specificity and low toxicity owing to their high binding affinity to their specific targets.

Two categories of currently available drugs exist, small molecules <500 Da active per os, and large molecules >5000 Da that are inefficient orally and need to be administered in the parenteral form as injections [START_REF] Craik | The future of peptide-based drugs[END_REF]. If small molecules are orally bioavailable, they could be designed with a lesser cost.

However, owing to their size, they could lack specificity and potentially could lead to off-target adverse effects. Large biologic drugs show high affinity for their targets but need to be injected into patients [START_REF] Craik | The future of peptide-based drugs[END_REF]. Spadin and its analogs belong to a class of molecules that range between small molecules and large polypeptides that represent a nonnegligible advantage for both drug administration and specificity for targets. Advantages of spadin and its analogs as promising AD drugs are summarized in Box 1. In addition to the central effect of spadin on depression through targeting TREK-1 channels in the brain, spadin diffuses in the blood circulation and reaches a number of organs including the pancreas and prostate. Indeed, as described above, in the pancreatic β-cells, spadin plays a crucial role as an insulin-releasing peptide upon hyperglycemia [START_REF] Hivelin | Potentiation of Calcium Influx and Insulin Secretion in Pancreatic Beta Cell by the Specific TREK-1 Blocker Spadin[END_REF].

More than 60 peptides have been approved by the US Food and Drug Administration (FDA), and this is expected to greatly increase in the near future, as more than 140 molecules are currently being processed and tested in clinical trials. In addition, at least 500 peptides are under investigation in preclinical studies [START_REF] Fosgerau | Peptide therapeutics: current status and future directions[END_REF][START_REF] Kaspar | Future directions for peptide therapeutics development[END_REF] and are quite ready to be tested in humans.

Spadin and its analogs undoubtedly constitute important candidate peptides intended to treat depression in clinic. As natural peptides, they irrefutably represent a huge advantage compared to small classical ADs, as the risk-benefit ratio will be sharply improved. The next step consists of validating their efficacy and tolerability processes in healthy volunteers during clinical trials. Many peptides have been designed for various pathologies such as cancer, diabetes, and infectious diseases. However, only few peptides are used in the treatment of neurological disorders; this is mainly due to their weak BBB crossing and their rapid degradation in vivo [START_REF] Mcgowan | Challenges and new strategies for therapeutic peptide delivery to the CNS[END_REF].

These observations are not applicable to spadin because the effects of peripheral i.p. injections of spadin and its analogs (at doses as low as 100 µg/kg) can be measured in the brain [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. Additionally, the in vivo stability of spadin ranges between 7 and 23 h, a duration that is compatible with the use in human clinic. As the validation of TREK-1 as a potential target in the treatment of depression, TREK-1 druggability was made possible after the discovery of spadin and its analogs (Fig. 4).

Thereafter, spadin efficacy was gradually optimized through different strategies such as RI strategy, cyclization, sequence shortening, and modifications of N-and Cterminal ends. Ultimately, efficiency, inhibition potency, and in vivo stability of these analogs are significantly improved compared to spadin. Moreover, AD activity is maintained despite shortening and modifying the peptide sequence. Interestingly, the AD phenotype is associated with an enhancement of both in vivo hippocampal neurogenesis and cortical synaptogenesis. It will be challenging to overcome obstacles and therefore lead spadin analogs to the finish line, i.e., marketing spadin analogs as novel AD drugs with an original mechanism of action. However, all these observations give rise to a very positive signal for the use of spadin and/or its derivatives as new ADs. Nevertheless, we have to keep in mind that all these preclinical studies were performed on animal models described to mimic a part of a clinical symptom of depression. Definitive answers can be obtained from clinical trials.

To our knowledge, spadin and its analogs are the first therapeutic peptides with a high potential to successfully go through clinical trials and thus be marketed for the treatment of mood disorders. Methods. hTREK-1/HEK cells were seeded at a density of 20,000 cells/35 mm dish.

Electrophysiological recordings were performed 24-48h after plating using the wholecell configuration of patch-clamp technique. In order to measure TREK-1 current (ITREK-1), a cocktail of potassium channel blockers was added to the bath solution. This cocktail contained: 3 mM 4-AP (4-aminopyridine), 10 mM TEA (tetraethylammonium), 10 µM Glibenclamide, 100 nM Apamin and 50 nM Charybdotoxin (for more experimental details see [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]). Whole-cell ITREK-1 currents were generated by running a pulse or ramp protocol every 5 s from -100 to +60 mV with a holding potential maintained at -80 mV. The inhibitory effect on TREK-1 channels of 100nM of spadin analogs were compared with the inhibitory effect of spadin, on cells pre-activated with 10 µM of arachidonic acid (AA). Patchclamp recording data were analyzed using Clampfit (Molecular Devices, USA). I=f(V) curves were obtained from -100 to +60 mV ramp. Data were presented as mean ± SEM from at least 3 independent experiments. In GraphPad Prism (GraphPad software, La Jolla, USA), statistical comparisons were performed using Student's ttest or ANOVA one-way. A result is considered as statistically significant when p< 0.05. In the FST, when administered intravenously, analog 13 and analog 14 significantly decreased the immobility time in mice compared to saline injected mice (control).

However, analog 15 and analog 16 were unable to display AD-like behavior when i.v injected. b. Intracerebroventricular route (i.c.v) demonstrated that analog 15 did not cross the BBB while analog 16 remains inefficient in reducing the immobility time in mice. *, p<0.05; **, p<0.01; ns, not significant.

Methods. 30 min before the test, animals were injected with the different spadin's analogs by using either the intraveinous route (iv) or the intracerebroventricular route (icv). Mice were individually placed for 6 minutes in a non-escapable cylinder (30 cm height and 15 cm diameter) half-filled with water at 22°C ± 1°C. The immobility time was manually measured only during the last 4 minutes. A mouse was considered immobile when it remained immobile with only slight movements in order to keep its head above water [START_REF] Porsolt | Depression: a new animal model sensitive to antidepressant treatments[END_REF].

Statistical comparisons were performed using ANOVA one-way. FST (acute and chronic -14-21 days) no effect after 3 day subchronic treatment slow 2-4 weeks [START_REF] Detke | Active behaviors in the rat forced swimming test differentially produced by serotonergic and noradrenergic antidepressants[END_REF][START_REF] Kennard | Inhibition of the human two-pore domain potassium channel, TREK-1, by fluoxetine and its metabolite norfluoxetine[END_REF][START_REF] Kim | Activation of TREK-1, but Not TREK-2, Channel by Mood Stabilizers[END_REF][START_REF] Wong | A selective inhibitor of serotonin uptake: Lilly 110140, 3-(p-trifluoromethylphenoxy)-Nmethyl-3-phenylpropylamine[END_REF] norfluoxetine HEK 293 9 µM blocks TREK-2 (IC50=4.9±0.5 µM) nd nd [START_REF] Kim | Activation of TREK-1, but Not TREK-2, Channel by Mood Stabilizers[END_REF][START_REF] Kobayashi | Inhibition of G protein-activated inwardly rectifying K+ channels by the antidepressant paroxetine[END_REF][START_REF] Lee | Antidepressant drug paroxetine blocks the open pore of Kv3.1 potassium channel[END_REF][START_REF] Mcclenaghan | Polymodal activation of the TREK-2 K2P channel produces structurally distinct open states[END_REF][START_REF] Thummler | Antipsychotics inhibit TREK but not TRAAK channels[END_REF] paroxetine

Spadin and analogs

spadin (PE 12-28) Cos-7, HEK293 40 nM specific to TREK-1 no effect on TREK-2, TRAAK, TRESK, TASK-1, and hERG channels FST (acute and subchronic), TST, LHT, and NSF (subchronic)

fast only after 4 days [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF][START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF] PE 22-28 HEK 293 0.12 nM [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF] G

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Spadin analogs display strong TREK-1 inhibition. Spadin analogs

Figure 3 .

 3 Figure 3. Antidepressant activity of spadin analogs in the Forced Swim Test. a.

Figure 4 .

 4 Figure 4. Schematic representation of spadin development as new concept in

Table 2 TABLE 1 :

 21 Molecules described as TREK-1 blockers.

	TREK-1 blockers	Cell types	IC50	specificity	Antidepressant activity	Onset of action	Reference
	fluoxetine	HEK 293	19 µM 37.9±7.7 µM	blocks TREK-2 (IC50=28.7±7.6 µM), SERT and TASK-3
	SSRIs

TABLE 2 :

 2 Summary of effects of spadin analogs on TREK-1 inhibition and FST immobility time.

	Peptide	Peptide names Modifications	% of	Significance FST	Significance
	number			TREK-1		in
				inhibition		acute
						(s)
	1	PE 12-28 (Spadin)	No modification	87.08 ± 7.32	***	5.05 107.40 ±	***
	2	Ac-PE 12-28	N-acetylation	83.46 ± 8.33	***	nd	nd
	3	Ac-RI-PE 12-28	spadin retro-inverso of	94.43 ± 10.87	***	8.11 135.10 ±	**
	4	Ac-PE-22-28	N-acetylation	19.29 ± 25.7	ns	nd	nd
	5	Ac-PE-21-28	N-acetylation	26.89 ± 14.9	ns	nd	nd
	6	Ac-RI-PE-21-28	retro-inverso N-acetylation +	31.19 ± 17.8	ns	nd	nd
	7	Ac-PE 1-28	N-acetylation	10.43 ± 28.5	ns	nd	nd
	8	Ac-RI-PE 1-28	retro-inverso N-acetylation +	108.59 ± 10	***	9.01 83.60 ±	***
	9	Ac-PE 6-28	N-acetylation	71.85 ± 28.12	**	nd	nd
	10	Ac-RI-PE 6-28	retro-inverso N-acetylation +	56.47 ± 24.13	*	nd	nd
	11	Ac-PE 1-44	N-acetylation	26.97 ± 6.25	ns	nd	nd
	12	Ac-RI-PE 1-44	retro-inverso N-acetylation +	37.73 ± 8.77	ns	nd	nd
	13	G/A-PE 12-28	aa substitution	83.5 ± 9.76	***	9.7 120.4 ±	**
	14	RI-G/A-PE 12-28	substitution retro-inverso +aa	72.15 ± 11.75	***	8.32 127.7 ±	**
	15	c(RI-PE 12-28)	cyclization	67.24 ± 3.41	***	8.09 157 ±	ns
	16	c(RI-PE 12-28)2	cyclization	91.25 ± 8.14	***	12.99 146.7 ±	ns
	17	PE 12-27	No modification	28.39 ± 9.916	**	5.0 100.2 ±	***
	18	PE 14-25	No modification	0	ns	7.1 112.2 ±	**
	19	PE 22-27	No modification	25.7 ± 20.01	ns	4.2 168.2 ±	ns
	20	PE 22-25	No modification	36.02 ± 17.47	*	5.0 100.2 ±	***
	21	PE 22-28	No modification	55.46 ± 4.555	***	6.1 91.8 ±	***
	22	Biotin-PE 22-28	N-biotinylation	53.03 ± 6.416	***	4.3 112.1 ±	***
	23	Dansyl-PE 22-28	N-dansylation	48.78 ± 14.52	**	11.8 104.6 ±	***
	24	PE 22-28-O-Methyl	C-methoxylation	42.98 ± 13.47	**	8.1 137.1 ±	*
	25	PE 22-28-O-Ethyl	C-ethoxylation	41.39 ± 11.52	**	8.5 113.2 ±	***
	26	Formyl-PE 22-28	N-formylation	32.45 ± 12.22	*	nd	nd
	27	G/A-PE 22-28	aa substitution	50.61 ± 7.935	***	3.6 110.2 ±	***
	28	Biotin-G/A-PE 22-28	biotinylation aa substitution+N-	46.11 ± 7.743	***	7.1 140.7 ±	*
	29	PI-PE 22-28	No modification	46.19 ± 7.565	***	11.8 119.7 ±	**
	30	Biotin-PI-PE 22-28	N-biotinylation	49.11 ± 7.454	***	11.7 124.1 ±	**
	31	Palmitoyl-PE 22-28	N-palmitoylation	26.69 ± 16.45	ns	nd	nd
	32	FITC-PE 22-28	N-FITC group	22.1 ± 12.63	ns	nd	nd

Acknowledgments

We thank 3P3D startup, and particularly its director Dr Serge Richard for his friendly support, confidence in the spadin project, and fruitful discussions. We thank Franck Aguila for artwork contribution. We particularly thank Dr A. Patel and S. Morgan for careful reading of the manuscript.

Funding sources

This work was supported by the Centre National de la Recherche Scientifique and the Agence Nationale de la Recherche (ANR-13-SAMA-0001 and 0002 and ANR-13-RPIB-0001 and 0002). We also thank the French Government for the "Investments for the Future" LabEx ICST #ANR-11 LabEx 0015 and the Fondation de l'Avenir No.

AP-RMA-2015-021. M. Pietri was supported by a CIFRE fellowship.

Abbreviations

Conflict of Interest Statement

The authors declare that they are no conflicts of interest.

Authorship Contributions

AD wrote the manuscript. CH, JM, and MB corrected and improved the manuscript.

AD conducted patch-clamp experiments, and MP and MB performed behavioral tests. CH, JM, and MB contributed to reagents/materials/analysis tools.

Box 1

Potential advantages of spadin and its analogs as antidepressant molecules

Fast acting. Psychiatrists, physicians, and patients want drugs that act more rapidly than those that are prescribed presently. Current marketed AD drugs often take several weeks to be efficient. Spadin or its analogs only need 4 days to exert their AD effects [START_REF] Mazella | Spadin, a sortilin-derived peptide, targeting rodent TREK-1 channels: a new concept in the antidepressant drug design[END_REF]. Additionally, spadin and its analogs are effective regardless of the route they are administered: intravenous, intraperitoneal, intracerebroventricular, subcutaneous, or per os.

Specificity. Spadin and its analogs are highly specific for TREK-1 channels. They do not modify the activity of other K2P channels such as TREK-2, TRAAK, TRESK, and TASK 1 channels [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF][START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF]. This is particularly remarkable because TREK-2 and TRAAK belong to the same subfamily and share almost 80% homologies with TREK-1 [START_REF] Noel | Molecular regulations governing TREK and TRAAK channel functions[END_REF].

Cardiac safety. In rodent, spadin and its analogs do not affect hERG or KCNQ1/KCNE1 channels and do not modify heart rates. These data indicate that spadin and its analogs have no deleterious effects at the cardiac level [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF][START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF].

No side effects on TREK-1-controlled functions. In addition to the absence of effects on the heart, preclinical studies have shown that spadin and its analogs have no deleterious side effects on pathologies that are under the control of TREK-1 channels, such as pain, epilepsy, or ischemia [START_REF] Ou Maati | Spadin as a new antidepressant: absence of TREK-1-related side effects[END_REF].

High efficiency. The development of spadin analogs has increased the AD potency of the original peptide. Analogs of concentration 3 µg/kg are adequate to produce an AD effect same as that obtained with a concentration of 100 µg/kg [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF].

Natural peptides. In addition to the fact that they are natural peptides, we can reasonably expect a high level of specificity and safety for these peptides. Converse to the majority of actual ADs that provoke withdrawal behavior, the probability to observe such a phenomenon with a natural peptide is low.

Duration effects. The relatively short time (7 hours) of in vivo effects of spadin could have been a problem, but it was solved with the development of spadin analogs. Therefore, the in vivo stability has become very close to 24 hours [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]. This sustained effect is important for use in clinics.

Biomarker. Spadin-like activity in the serum, mainly corresponding to PE and its degradation products, is significantly decreased in patients suffering from MDD in comparison to healthy controls [START_REF] Devader | Serum sortilin-derived propeptides concentrations are decreased in major depressive disorder patients[END_REF].

Comparison with other putative drugs. Other TREK-1 blockers such as SID1900 or lig4-4 have been recently identified. Although these molecules were described to be specific blockers of TREK-1 channels, they probably affect other types of ion channels as indicated in Table 1. Furthermore, they show very low affinities for TREK-1 channels [START_REF] Wang | Lig4-4 selectively inhibits TREK-1 and plays potent neuroprotective roles in vitro and in rat MCAO model[END_REF][START_REF] Ye | TREK1 channel blockade induces an antidepressant-like response synergizing with 5-HT1A receptor signaling[END_REF] in comparison to spadin or its analogs (several µM vs. nM) [START_REF] Djillani | Shortened Spadin Analogs Display Better TREK-1 Inhibition, In Vivo Stability and Antidepressant Activity[END_REF]. Many other molecules are able to block TREK-1 channels, but none of them is specific for TREK-1. Nevertheless, these molecules were not extensively studied in the field of depression, and their properties are detailed in the paragraph 2.3.2 of this review. Some microRNAs were also supposed to be involved in the depression process, but it is very soon to really define a therapeutic strategy using microRNAs [START_REF] Yuan | Circulating microRNAs as biomarkers for depression: Many candidates, few finalists[END_REF]. Recently, ketamine was also described [START_REF] Ramaker | Identifying fast-onset antidepressants using rodent models[END_REF] as a fast-acting molecule, but ketamine needs to be frequently administered (sometimes in hours) to obtain a sustained effect [START_REF] Schwartz | Ketamine for treatment-resistant depression: recent developments and clinical applications[END_REF]. This point is an important drawback in the use of ketamine. Additionally, ketamine displays numerous adverse effects [START_REF] Li | Ketamine: 50 Years of Modulating the Mind[END_REF].