In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin.

BACKGROUND AND PURPOSE: We have described a novel antidepressant peptide, spadin, that acts by blocking the TWIK-related-potassium channel, type 1 (TREK-1). Here, we examined possible mechanisms of action of spadin at both molecular and cellular levels. EXPERIMENTAL APPROACHES: Effects of spadin were measured in primary cultures of neurons or tissues from mice injected i.v. with spadin. Western blots, qPCR, histochemical and electrophysiological techniques were used. KEY RESULTS: In vitro, spadin increased neuronal membrane potential and activated both the MAPK and PI3K signalling pathways, in a time- and concentration-dependent manner. The latter pathway was involved in the protective effect of spadin against staurosporine-induced apoptosis. Also, spadin enhanced both mRNA expression and protein of two markers of synaptogenesis, the post-synaptic density protein of 95 kDalton (PSD-95) and synapsin. We confirmed these effects on synaptogenesis by the observation that spadin treatment significantly increased the proportion of mature spines in cortical neurons. Finally, in vivo injections of spadin led to a rapid increase in both mRNA expression and protein level of brain-derived neurotrophic factor (BDNF) in the hippocampus, confirming the antidepressant action of the peptide. We argue for a new role of spadin in synaptogenesis as both PSD-95 and synapsin mRNA expression and protein levels were further enhanced in the hippocampus, following treatment in vivo with the peptide. CONCLUSIONS AND IMPLICATIONS: These findings provide new mechanisms of action for the rapidly acting antidepressant peptide spadin by stimulating expression of BDNF and synaptic proteins, both in vitro and in vivo.

Production, in Pichia pastoris, of a recombinant monomeric mapacalcine, a protein with anti-ischemic properties.

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.

Targeting two-pore domain K(+) channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept.

Depression is a disease that is particularly frequent, affecting up to 20% of the population in Western countries. The origins of this pathology involve multiple genes as well as environmental and developmental factors leading to a disorder that remains difficult to treat. Several therapies for depression have been developed and these mainly target monoamine neurotransmitters. However, these treatments are not only associated with numerous adverse effects, but they are also ineffective for more than one-third of patients. Therefore, the need to develop new concepts to treat depression is crucial. Recently, studies using knockout mouse models have provided evidence for a crucial role of two members of the two-pore domain potassium channel (K2P ) family, tandem P-domain weak inward rectifying K(+) (TWIK)-related K(+) channel 1 (TREK-1) and TWIK-related acid-sensitive K(+) channel 3 (TASK-3) in the pathophysiology of depression. It is believed that TREK-1 and TASK-3 antagonists could lead to the development of new antidepressants. Herein, we describe the discovery of spadin, a natural peptide released from the maturation of the neurotensin receptor-3 (also known as sortilin), which specifically blocks the activity of the TREK-1 channel and displays particular antidepressant properties, with a rapid onset of action and the absence of adverse effects. The development of such molecules may open a new era in the field of psychiatry.

NeuroAiD: properties for neuroprotection and neurorepair.

BACKGROUND: Treatments for stroke and other brain injuries are limited. NeuroAiD has been shown to be beneficial in clinical studies. We reviewed the pharmacological effects of NeuroAiD on the normal and ischemic brain and neurons. METHODS: In vivo and in vitro experiments using mouse model of stroke (focal ischemia), rat model of cardiac arrest (global ischemia) and cortical neurons in culture were reviewed and summarized. RESULTS: NeuroAiD improved survival, attenuated infarct size, improved functional recovery in the model of focal ischemia, and protected neurons against glutamate-induced injury. Furthermore, it enhanced cognitive recovery by reducing hippocampal CA1 cell degeneration, DNA fragmentation, Bax expression and ma-londialdehyde release in the model of global ischemia. Activation of the Akt survival pathway and opening of KATP channels may contribute to the neuroprotective properties of NeuroAiD. NeuroAiD increased BDNF expression and induced proliferation of cells which differentiate and mature into neurons. It enhanced rosette formation of human embryonic stem cells. NeuroAiD-treated embryonic cortical neurons developed into neurons with longer neurites, denser outgrowths and networks, and more synaptic release sites. CONCLUSIONS: NeuroAiD demonstrated both neuroprotective and neuroregenerative properties in rodent models of focal and global ischemia and in cortical cell cultures. These properties would be important for developing a treatment strategy in reducing the long-term disability of stroke, cardiac arrest and other brain injuries.

Activation of ATP-sensitive potassium channels as an element of the neuroprotective effects of the Traditional Chinese Medicine MLC901 against oxygen glucose deprivation.

NeuroAid (MLC601 and MLC901), a Traditional Medicine used in China for patients after stroke has been reported in preclinical models of ischemia to induce neuroprotection and neuroplasticity. This work shows the effects of MLC901 on an in vitro model of oxygen glucose deprivation (OGD). MLC901 prevents neuronal death induced by 120 min OGD and decreases the exaggerated Ca²âº entry in mature cortical neurons exposed to 120 min OGD. The neuroprotective effect of MLC901 is associated with a large hyperpolarization of ∼20 mV which is antagonized by glibenclamide, the specific inhibitor of K(ATP) channels. In addition MLC901 strengthens the activation of K(ATP) channels. MLC901 has been directly shown to act as an activator of K(ATP) channels as potent as the classical K(ATP) channel opener. The capacity of MLC901 to produce a large hyperpolarization, particularly in neurons that have suffered from energy deprivation probably plays an important role in the neuroprotective effects of this traditional medicine that comes in addition to its previously demonstrated neuroregenerative properties.

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon.

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 µM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 µM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 µM), ODQ (10 µM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 µM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 µM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 µM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.

Spadin as a new antidepressant: absence of TREK-1-related side effects.

Despite several decades of research, current antidepressant (AD) treatments remain of a limited efficacy justifying the need to find new drugs. These drugs have to be more efficacious, more rapid and display lesser side effects. Using rodent models, we recently identified spadin as a new antidepressant molecule that acts more quickly than classical ADs, working within 4 days to get same effects obtained with other ADs after 21 days. Spadin blocks TREK-1 K(2P) potassium channels that are considered as new targets for ADs. Deletion of the TREK-1 channel is known to increase sensitivity to pain, seizures and ischemia. Thus blocking these channels could result in deleterious side effects. In this study we showed that spadin did not interfere with other TREK-1 controlled functions such as pain, epilepsy and ischemia. We also demonstrated that spadin was unable to inhibit currents generated by TREK-2, TRAAK, TASK and TRESK four other K2P channels. More importantly, spadin did not induce cardiac dysfunctions, did not block I(Kr) and I(Ks) and did not modify the systolic pressure or cardiac pulses. After a three week treatment spadin remained an efficacious AD and did not modify the infarct size in brain following focal ischemia. Finally, we showed that kainate induced seizures and glycemia were not modified by spadin treatments. These data, together with those previously published reinforce the idea that spadin represents a good candidate for a new generation of ADs. This article is part of a Special Issue entitled 'Anxiety and Depression'.

Dexrazoxane protects the heart from acute doxorubicin-induced QT prolongation: a key role for I(Ks).

INTRODUCTION: Doxorubicin, an anthracycline widely used in the treatment of a broad range of tumours, causes acute QT prolongation. Dexrazoxane has been shown to prevent the QT prolongation induced by another anthracycline, epirubicin, but has not yet been reported to prevent that induced by doxorubicin. Thus, the present study was designed to test whether the acute QT effects induced by doxorubicin could be blocked by dexrazoxane and to explore the mechanism. Results were compared with those obtained with a reference human ether-a-go-go (hERG) channel blocker, moxifloxacin. METHODS: The effects of moxifloxacin (100 microM) and doxorubicin (30 microM), with or without dexrazoxane (from 3 to 30 microM), have been evaluated on the QTc interval in guinea-pig isolated hearts and on I(Kr) (rapid component of the delayed rectifier current) and I(Ks) (slow component of the delayed rectifier current) currents stably expressed in human embryonic kidney 293 cells. RESULTS: Moxifloxacin (100 microM), a potent hERG blocker, prolonged QTc by 22%, and this effect was not prevented by dexrazoxane. Doxorubicin (30 microM) also prolonged QTc by 13%, did not significantly block hERG channels and specifically inhibited I(Ks) (IC(50): 4.78 microM). Dexrazoxane significantly reduced the doxorubicin-induced QTc prolongation and prevented doxorubicin-induced inhibition of I(Ks). CONCLUSION AND IMPLICATIONS: Doxorubicin acutely prolonged the QT interval in guinea-pig heart by selective I(Ks) blockade. This effect was prevented by dexrazoxane. This result is important because it illustrates the danger of neglecting I(Ks) in favour of hERG screening alone, for early preclinical testing for possible induction of torsade de pointes.