Antidepressive effects of targeting ELK-1 signal transduction.

Depression, a devastating psychiatric disorder, is a leading cause of disability worldwide. Current antidepressants address specific symptoms of the disease, but there is vast room for improvement 1 . In this respect, new compounds that act beyond classical antidepressants to target signal transduction pathways governing synaptic plasticity and cellular resilience are highly warranted2-4. The extracellular signal-regulated kinase (ERK) pathway is implicated in mood regulation5-7, but its pleiotropic functions and lack of target specificity prohibit optimal drug development. Here, we identified the transcription factor ELK-1, an ERK downstream partner 8 , as a specific signaling module in the pathophysiology and treatment of depression that can be targeted independently of ERK. ELK1 mRNA was upregulated in postmortem hippocampal tissues from depressed suicides; in blood samples from depressed individuals, failure to reduce ELK1 expression was associated with resistance to treatment. In mice, hippocampal ELK-1 overexpression per se produced depressive behaviors; conversely, the selective inhibition of ELK-1 activation prevented depression-like molecular, plasticity and behavioral states induced by stress. Our work stresses the importance of target selectivity for a successful approach for signal-transduction-based antidepressants, singles out ELK-1 as a depression-relevant transducer downstream of ERK and brings proof-of-concept evidence for the druggability of ELK-1.

Stress hormone rapidly tunes synaptic NMDA receptor through membrane dynamics and mineralocorticoid signalling.

Stress hormones, such as corticosteroids, modulate the transmission of hippocampal glutamatergic synapses and NMDA receptor (NMDAR)-dependent synaptic plasticity, favouring salient behavioural responses to the environment. The corticosterone-induced synaptic adaptations partly rely on changes in NMDAR signalling, although the cellular pathway underlying this effect remains elusive. Here, we demonstrate, using single molecule imaging and electrophysiological approaches in hippocampal neurons, that corticosterone specifically controls GluN2B-NMDAR surface dynamics and synaptic content through mineralocorticoid signalling. Strikingly, extracellular corticosterone was sufficient to increase the trapping of GluN2B-NMDAR within synapses. Functionally, corticosterone-induced potentiation of AMPA receptor content in synapses required the changes in NMDAR surface dynamics. These high-resolution imaging data unveiled that, in hippocampal networks, corticosterone is a natural, potent, fast and specific regulator of GluN2B-NMDAR membrane trafficking, tuning NMDAR-dependent synaptic adaptations.

Hippocampal Fast Glutamatergic Transmission Is Transiently Regulated by Corticosterone Pulsatility.

In recent years it has become clear that corticosteroid hormones (such as corticosterone) are released in ultradian pulses as a natural consequence of pituitary-adrenal interactions. All organs, including the brain, are thus exposed to pulsatile changes in corticosteroid hormone level, important to ensure full genomic responsiveness to stress-induced surges. However, corticosterone also changes neuronal excitability through rapid non-genomic pathways, particularly in the hippocampus. Potentially, background excitability of hippocampal neurons could thus be changed by pulsatile exposure to corticosteroids. It is currently unknown, though, how neuronal activity alters during a sequence of corticosterone pulses. To test this, hippocampal cells were exposed in vitro to four consecutive corticosterone pulses with a 60 min inter-pulse interval. During the pulses we examined four features of hippocampal signal transfer by the main excitatory transmitter glutamate-i.e., postsynaptic responses to spontaneous release of presynaptic vesicles, postsynaptic GluA2-AMPA receptor dynamics, basal (evoked) field responses, and synaptic plasticity, using a set of high resolution imaging and electrophysiological approaches. We show that the first pulse of corticosterone causes a transient increase in miniature EPSC frequency, AMPA receptor trafficking and synaptic plasticity, while basal evoked field responses are unaffected. This pattern is not maintained during subsequent applications: responses become more variable, attenuate or even reverse over time, albeit with different kinetics for the various experimental endpoints. This may indicate that the beneficial effect of ultradian pulses on transcriptional regulation in the hippocampus is not consistently accompanied by short-term perturbations in background excitability. In general, this could be interpreted as a means to keep hippocampal neurons responsive to incoming signals related to environmental challenges.

Ultradian corticosterone pulses balance glutamatergic transmission and synaptic plasticity.

The rodent adrenal hormone corticosterone (CORT) reaches the brain in hourly ultradian pulses, with a steep rise in amplitude before awakening. The impact of a single CORT pulse on glutamatergic transmission is well documented, but it remains poorly understood how consecutive pulses impact on glutamate receptor trafficking and synaptic plasticity. By using high-resolution imaging and electrophysiological approaches, we report that a single pulse of CORT to hippocampal networks causes synaptic enrichment of glutamate receptors and increased responses to spontaneously released glutamatergic vesicles, collectively abrogating the ability to subsequently induce synaptic long-term potentiation. Strikingly, a second pulse of CORT one hour after the first--mimicking ultradian pulses--completely normalizes all aspects of glutamate transmission investigated, restoring the plastic range of the synapse. The effect of the second pulse is precisely timed and depends on a nongenomic glucocorticoid receptor-dependent pathway. This normalizing effect through a sequence of CORT pulses--as seen around awakening--may ensure that hippocampal glutamatergic synapses remain fully responsive and able to encode new stress-related information when daily activities start.

Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses.

NMDA-type glutamate receptors (NMDAR) are central actors in the plasticity of excitatory synapses. During adaptive processes, the number and composition of synaptic NMDAR can be rapidly modified, as in neonatal hippocampal synapses where a switch from predominant GluN2B- to GluN2A-containing receptors is observed after the induction of long-term potentiation (LTP). However, the cellular pathways by which surface NMDAR subtypes are dynamically regulated during activity-dependent synaptic adaptations remain poorly understood. Using a combination of high-resolution single nanoparticle imaging and electrophysiology, we show here that GluN2B-NMDAR are dynamically redistributed away from glutamate synapses through increased lateral diffusion during LTP in immature neurons. Strikingly, preventing this activity-dependent GluN2B-NMDAR surface redistribution through cross-linking, either with commercial or with autoimmune anti-NMDA antibodies from patient with neuropsychiatric symptoms, affects the dynamics and spine accumulation of CaMKII and impairs LTP. Interestingly, the same impairments are observed when expressing a mutant of GluN2B-NMDAR unable to bind CaMKII. We thus uncover a non-canonical mechanism by which GluN2B-NMDAR surface dynamics plays a critical role in the plasticity of maturing synapses through a direct interplay with CaMKII.

Disrupted surface cross-talk between NMDA and Ephrin-B2 receptors in anti-NMDA encephalitis.

Autoimmune synaptic encephalitides are recently described human brain diseases leading to psychiatric and neurological syndromes through inappropriate brain-autoantibody interactions. The most frequent synaptic autoimmune encephalitis is associated with autoantibodies against extracellular domains of the glutamatergic N-methyl-d-aspartate receptor, with patients developing psychotic and neurological symptoms in an autoantibody titre-dependent manner. Although N-methyl-d-aspartate receptors are the primary target of these antibodies, the cellular and molecular pathway(s) that rapidly lead to N-methyl-d-aspartate receptor dysfunction remain poorly understood. In this report, we used a unique combination of high-resolution nanoparticle and bulk live imaging approaches to demonstrate that anti-N-methyl-d-aspartate receptor autoantibodies from patients with encephalitis strongly alter, in a time-dependent manner, the surface content and trafficking of GluN2-NMDA receptor subtypes. Autoantibodies laterally displaced surface GluN2A-NMDA receptors out of synapses and completely blocked synaptic plasticity. This loss of extrasynaptic and synaptic N-methyl-d-aspartate receptor is prevented both in vitro and in vivo, by the activation of EPHB2 receptors. Indeed, the anti-N-methyl-d-aspartate receptor autoantibodies weaken the interaction between the extracellular domains of the N-methyl-d-aspartate and Ephrin-B2 receptors. Together, we demonstrate that the anti-N-methyl-d-aspartate receptor autoantibodies from patients with encephalitis alter the dynamic retention of synaptic N-methyl-d-aspartate receptor through extracellular domain-dependent mechanism(s), shedding new light on the pathology of the neurological and psychiatric disorders observed in these patients and opening possible new therapeutic strategies.

Dynamic and specific interaction between synaptic NR2-NMDA receptor and PDZ proteins.

The relative content of NR2 subunits in the NMDA receptor confers specific signaling properties and plasticity to synapses. However, the mechanisms that dynamically govern the retention of synaptic NMDARs, in particular 2A-NMDARs, remain poorly understood. Here, we investigate the dynamic interaction between NR2 C termini and proteins containing PSD-95/Discs-large/ZO-1 homology (PDZ) scaffold proteins at the single molecule level by using high-resolution imaging. We report that a biomimetic divalent competing ligand, mimicking the last 15 amino acids of NR2A C terminus, specifically and efficiently disrupts the interaction between 2A-NMDARs, but not 2B-NMDARs, and PDZ proteins on the time scale of minutes. Furthermore, displacing 2A-NMDARs out of synapses lead to a compensatory increase in synaptic NR2B-NMDARs, providing functional evidence that the anchoring mechanism of 2A- or 2B-NMDARs is different. These data reveal an unexpected role of the NR2 subunit divalent arrangement in providing specific anchoring within synapses, highlighting the need to study such dynamic interactions in native conditions.

Matrix metalloproteinase-9 controls NMDA receptor surface diffusion through integrin beta1 signaling.

Matrix metalloproteinase-9 (MMP-9) has emerged as a physiological regulator of NMDA receptor (NMDAR)-dependent synaptic plasticity and memory. The pathways by which MMP-9 affects NMDAR signaling remain, however, elusive. Using single quantum dot tracking, we demonstrate that MMP-9 enzymatic activity increases NR1-NMDAR surface trafficking but has no influence on AMPA receptor mobility. The mechanism of MMP-9 action on NMDAR is not mediated by change in overall extracellular matrix structure nor by direct cleavage of NMDAR subunits, but rather through an integrin beta1-dependent pathway. These findings describe a new target pathway for MMP-9 action in key physiological and pathological brain processes.

Altered surface trafficking of presynaptic cannabinoid type 1 receptor in and out synaptic terminals parallels receptor desensitization.

Presynaptic cannabinoid type 1 receptors (CB1Rs) are major mediators of retrograde synaptic plasticity at both excitatory and inhibitory synapses and participate in a plethora of physiological functions. Whether presynaptic receptors, such as CB1R, display functionally relevant movements at the surface of neuronal membranes is not known. We analyzed the lateral mobility of native CB1Rs in cortical neurons by using single-quantum dot imaging. We found that CB1Rs are highly mobile and rapidly diffuse in and out of presynapses. Agonist-induced desensitization correlated with a reduction in the fraction of surface CB1Rs and a drastic decrease in the membrane dynamic of the CB1Rs that remained at the presynaptic surface. Desensitization specifically excluded CB1Rs from synapses and increased the fraction of immobile receptors in the extrasynaptic compartment. The results suggest that decrease of mobility may be one of the core mechanisms underlying the desensitization of CB1R, the most abundant G protein-coupled receptor in the brain.

Chronic treatment with amyloid beta(1-42) inhibits non-cholinergic high-affinity choline transport in NG108-15 cells through protein kinase C signaling.

We investigated the influence of the amyloid-beta-peptide(1-42) on hemicholinum-3-sensitive high-affinity choline uptake in NG108-15 cells. RT-PCR analysis revealed the presence of mRNA for a choline transporter-like protein but not for cholinergic high-affinity choline transporter. Differentiation of cells increased both hemicholinum-3-sensitive choline uptake and high-affinity hemicholinium-3 binding. This transport was not influenced by tenfold excess of carnitine. Continuous presence of submicromolar concentrations of amyloid-beta-peptide(1-42) during differentiation resulted in a decrease of both choline uptake and hemicholinium-3 binding. These effects were not present when amyloid-beta-peptide(1-42) was added 5 min prior to measurements. Neither differentiation nor amyloid-beta-peptide(1-42) treatment changed levels of choline transporter-like protein mRNA. Protein kinase C inhibition by staurosporine or its inactivation by continuous presence of tetradecanoyl phorbol acetate prevented the inhibitory effect of amyloid-beta-peptide(1-42) treatment on choline uptake. Activation of protein kinase C by tetradecanoyl phorbol acetate during measurement had inhibitory effect on choline uptake in control but not amyloid-beta-peptide(1-42)-treated cells. The concentration of amyloid-beta-peptide(1-42) maximally effective on hemicholinium-3-sensitive choline uptake had no effect on cell growth, oxidative activity, membrane integrity, number of surface muscarinic receptors, caspase-3 and -8 activities, or uptake of deoxyglucose. Results demonstrate that long-term treatment with non-toxic concentrations of amyloid-beta-peptide(1-42) downregulates choline uptake presumably mediated by a choline transporter-like protein through activation of protein kinase C signaling. The decrease of choline uptake may have relevance to the pathogenesis of Alzheimer's disease.