Elena Nosyreva

NMDA receptor blockade at rest triggers rapid behavioural antidepressant responses

Clinical studies consistently demonstrate that a single sub-psychomimetic dose of ketamine, an ionotropic glutamatergic NMDAR (N-methyl-D-aspartate receptor) antagonist, produces fast-acting antidepressant responses in patients suffering from major depressive disorder, although the underlying mechanism is unclear. Depressed patients report the alleviation of major depressive disorder symptoms within two hours of a single, low-dose intravenous infusion of ketamine, with effects lasting up to two weeks, unlike traditional antidepressants (serotonin re-uptake inhibitors), which take weeks to reach efficacy. This delay is a major drawback to current therapies for major depressive disorder and faster-acting antidepressants are needed, particularly for suicide-risk patients. The ability of ketamine to produce rapidly acting, long-lasting antidepressant responses in depressed patients provides a unique opportunity to investigate underlying cellular mechanisms. Here we show that ketamine and other NMDAR antagonists produce fast-acting behavioural antidepressant-like effects in mouse models, and that these effects depend on the rapid synthesis of brain-derived neurotrophic factor. We find that the ketamine-mediated blockade of NMDAR at rest deactivates eukaryotic elongation factor 2 (eEF2) kinase (also called CaMKIII), resulting in reduced eEF2 phosphorylation and de-suppression of translation of brain-derived neurotrophic factor. Furthermore, we find that inhibitors of eEF2 kinase induce fast-acting behavioural antidepressant-like effects. Our findings indicate that the regulation of protein synthesis by spontaneous neurotransmission may serve as a viable therapeutic target for the development of fast-acting antidepressants.

Rapid Translation of Arc/Arg3.1 Selectively Mediates mGluR-Dependent LTD through Persistent Increases in AMPAR Endocytosis Rate

Salient stimuli that modify behavior induce transcription of activity-regulated cytoskeleton-associated protein (Arc/Arg3.1) and transport Arc mRNA into dendrites, suggesting that local Arc translation mediates synaptic plasticity that encodes such stimuli. Here, we demonstrate that long-term synaptic depression (LTD) in hippocampal neurons induced by group 1 metabotropic glutamate receptors (mGluRs) relies on rapid translation of Arc. mGluR-LTD induction causes long-term increases in AMPA receptor endocytosis rate and dendritic synthesis of Arc, a component of the AMPAR endocytosis machinery. Knockdown of Arc prevents mGluRs from triggering AMPAR endocytosis or LTD, and acute blockade of new Arc synthesis with antisense oligonucleotides blocks mGluR-LTD and AMPAR trafficking. In contrast, LTD induced by NMDA receptors does not persistently alter AMPAR endocytosis rate, induce Arc synthesis, or require Arc protein. These data demonstrate a role for local Arc synthesis specifically in mGluR-LTD and suggest that mGluR-LTD may be one consequence of Arc mRNA induction during experience.

Developmental Switch in Synaptic Mechanisms of Hippocampal Metabotropic Glutamate Receptor-Dependent Long-Term Depression

The presynaptic and postsynaptic properties of synapses change over the course of postnatal development. Therefore, synaptic plasticity mechanisms would be expected to adapt to these changes to facilitate alterations of synaptic strength throughout ontogeny. Here, we identified developmental changes in long-term depression (LTD) mediated by group 1 metabotropic glutamate receptors (mGluRs) and dendritic protein synthesis in hippocampal CA1 slices (mGluR-LTD). In slices prepared from adolescent rats [postnatal day 21 (P21) to P35], mGluR activation induces LTD and a long-term decrease in AMPA receptor (AMPAR) surface expression, both of which require protein synthesis. In neonatal animals (P8-P15), mGluR-LTD is independent of protein synthesis and is not associated with changes in the surface expression of AMPARs. Instead, mGluR-LTD at neonatal synapses results in large decreases in presynaptic function, measured by changes in paired-pulse facilitation and the rate of blockade by the use-dependent NMDA receptor blocker (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate. Conversely, mGluR-LTD at mature synapses results in little or no change in presynaptic function, suggesting a postsynaptic mechanism of expression. The developmental switch in the synaptic mechanisms of LTD would differentially affect synapse dynamics and perhaps information processing over the course of postnatal development.

Deletion of CASK in mice is lethal and impairs synaptic function

CASK is an evolutionarily conserved multidomain protein composed of an N-terminal Ca2+/calmodulin-kinase domain, central PDZ and SH3 domains, and a C-terminal guanylate kinase domain. Many potential activities for CASK have been suggested, including functions in scaffolding the synapse, in organizing ion channels, and in regulating neuronal gene transcription. To better define the physiological importance of CASK, we have now analyzed CASK “knockdown” mice in which CASK expression was suppressed by ≈70%, and CASK knockout (KO) mice, in which CASK expression was abolished. CASK knockdown mice are viable but smaller than WT mice, whereas CASK KO mice die at first day after birth. CASK KO mice exhibit no major developmental abnormalities apart from a partially penetrant cleft palate syndrome. In CASK-deficient neurons, the levels of the CASK-interacting proteins Mints, Veli/Mals, and neurexins are decreased, whereas the level of neuroligin 1 (which binds to neurexins that in turn bind to CASK) is increased. Neurons lacking CASK display overall normal electrical properties and form ultrastructurally normal synapses. However, glutamatergic spontaneous synaptic release events are increased, and GABAergic synaptic release events are decreased in CASK-deficient neurons. In contrast to spontaneous neurotransmitter release, evoked release exhibited no major changes. Our data suggest that CASK, the only member of the membrane-associated guanylate kinase protein family that contains a Ca2+/calmodulin-dependent kinase domain, is required for mouse survival and performs a selectively essential function without being in itself required for core activities of neurons, such as membrane excitability, Ca2+-triggered presynaptic release, or postsynaptic receptor functions.

Acute Suppression of Spontaneous Neurotransmission Drives Synaptic Potentiation

The impact of spontaneous neurotransmission on neuronal plasticity remains poorly understood. Here, we show that acute suppression of spontaneous NMDA receptor-mediated (NMDAR-mediated) neurotransmission potentiates synaptic responses in the CA1 regions of rat and mouse hippocampus. This potentiation requires protein synthesis, brain-derived neurotrophic factor expression, eukaryotic elongation factor-2 kinase function, and increased surface expression of AMPA receptors. Our behavioral studies link this same synaptic signaling pathway to the fast-acting antidepressant responses elicited by ketamine. We also show that selective neurotransmitter depletion from spontaneously recycling vesicles triggers synaptic potentiation via the same pathway as NMDAR blockade, demonstrating that presynaptic impairment of spontaneous release, without manipulation of evoked neurotransmission, is sufficient to elicit postsynaptic plasticity. These findings uncover an unexpectedly dynamic impact of spontaneous glutamate release on synaptic efficacy and provide new insight into a key synaptic substrate for rapid antidepressant action.

A Mouse Model of the Human Fragile X Syndrome I304N Mutation

The mental retardation, autistic features, and behavioral abnormalities characteristic of the Fragile X mental retardation syndrome result from the loss of function of the RNA–binding protein FMRP. The disease is usually caused by a triplet repeat expansion in the 5′UTR of the FMR1 gene. This leads to loss of function through transcriptional gene silencing, pointing to a key function for FMRP, but precluding genetic identification of critical activities within the protein. Moreover, antisense transcripts (FMR4, ASFMR1) in the same locus have been reported to be silenced by the repeat expansion. Missense mutations offer one means of confirming a central role for FMRP in the disease, but to date, only a single such patient has been described. This patient harbors an isoleucine to asparagine mutation (I304N) in the second FMRP KH-type RNA–binding domain, however, this single case report was complicated because the patient harbored a superimposed familial liver disease. To address these issues, we have generated a new Fragile X Syndrome mouse model in which the endogenous Fmr1 gene harbors the I304N mutation. These mice phenocopy the symptoms of Fragile X Syndrome in the existing Fmr1–null mouse, as assessed by testicular size, behavioral phenotyping, and electrophysiological assays of synaptic plasticity. I304N FMRP retains some functions, but has specifically lost RNA binding and polyribosome association; moreover, levels of the mutant protein are markedly reduced in the brain specifically at a time when synapses are forming postnatally. These data suggest that loss of FMRP function, particularly in KH2-mediated RNA binding and in synaptic plasticity, play critical roles in pathogenesis of the Fragile X Syndrome and establish a new model for studying the disorder.

Spontaneous Neurotransmission: An Independent Pathway for Neuronal Signaling?

Recent findings suggest that spontaneous neurotransmission is a bona fide pathway for interneuronal signaling that operates independent of evoked transmission via distinct presynaptic as well as postsynaptic substrates. This article will examine the role of spontaneous release events in neuronal signaling by focusing on aspects that distinguish this process from evoked neurotransmission, and evaluate the mechanisms that may underlie this segregation.

Functional and Biochemical Analysis of the Type 1 Inositol (1,4,5)-Trisphosphate Receptor Calcium Sensor

Modulation of the type 1 inositol (1,4,5)-trisphosphate receptors (InsP(3)R1) by cytosolic calcium (Ca(2+)) plays an essential role in their signaling function, but structural determinants and mechanisms responsible for the InsP(3)R1 regulation by Ca(2+) are poorly understood. Using DT40 cell expression system and Ca(2+) imaging assay, in our previous study we identified a critical role of E2100 residue in the InsP(3)R1 modulation by Ca(2+). By using intrinsic tryptophan fluorescence measurements in the present study we determined that the putative InsP(3)R1 Ca(2+)-sensor region (E1932-R2270) binds Ca(2+) with 0.16 micro M affinity. We further established that E2100D and E2100Q mutations decrease Ca(2+)-binding affinity of the putative InsP(3)R1 Ca(2+)-sensor region to 1 micro M. In planar lipid bilayer experiments with recombinant InsP(3)R1 expressed in Spodoptera frugiperda cells we discovered that E2100D and E2100Q mutations shifted the peak of the InsP(3)R1 bell-shaped Ca(2+) dependence from 0.2 micro M to 1.5 micro M Ca(2+). In agreement with the biochemical data, we found that the apparent affinities of Ca(2+) activating and inhibitory sites of the InsP(3)R1 were 0.2 micro M for the wild-type channels and 1-2 micro M Ca(2+) for the E2100D and E2100Q mutants. The results obtained in our study support the hypothesis that E2100 residue forms a part of the InsP(3)R1 Ca(2+) sensor.

Effects of a ketamine metabolite on synaptic NMDAR function

Clinical data have demonstrated rapid and sustained antidepressant effects of ketamine, a noncompetitive NMDAR (N-methyl-daspartate receptor) antagonist1. Recently, Zanos et al.2 claimed that the ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) is essential for the antidepressant effects of ketamine in mice in an NMDAR-independent manner, although no alternative mechanism was proposed, beyond unspecific activation of AMPAR (α -amino-3hydroxy-5-methyl-4-isoxazole propionic acid receptor)2. Here we report that (2R,6R)-HNK blocks synaptic NMDARs in a simi lar manner to its parent compound, and we show that the effects of (2R,6R)-HNK on intracellular signalling are coupled to NMDAR inhibition. These data demonstrate that (2R,6R)-HNK inhibits synaptic NMDARs and subsequently elicits the same signal transduction pathway previously associated with NMDAR inhibition by ketamine. There is a Reply to this Comment by Zanos, P. et al. Nature 546, http://dx.doi.org/10.1038/nature22085 (2017). In previous work3, we showed that ketamine exerts its antidepressant effects by blocking NMDARs at rest, which deactivates eukaryotic elongation factor 2 kinase (eEF2K), thereby dephosphorylating eukaryotic elongation factor 2 (eEF2) and resulting in a subsequent desuppression of brain-derived neurotrophic factor (BDNF) protein translation. This signalling pathway then potentiates synaptic AMPAR responses in the hippocampus through insertion of GluA1 and GluA2 subunits3–5. Notably, Zanos et al. show that (2R,6R)-HNK triggers the same intracellular pathway and downstream effects that we demonstrated for ketamine, namely inhibition of eEF2K, increased expression of BDNF, GluA1 and GluA2, and a form of synaptic potentiation in the hippocampus that is sensitive to AMPAR blockers3–5. The similarity between the molecular findings of ketamine and (2R,6R)-HNK led us to re-examine the potential involvement of (2R,6R)-HNK in NMDAR function. We assessed the effects of (2R,6R)-HNK in NMDAR-mediated miniature excitatory postsynaptic currents (NMDAR-mEPSCs) in cultured hippocampal neurons and compared its properties to the NMDAR antagonists 2R-amino-5-phosphonopentanoate (AP5) and ketamine. NMDA-mEPSCs were isolated in the presence of

Activity-Dependent Augmentation of Spontaneous Neurotransmission during Endoplasmic Reticulum Stress

The endoplasmic reticulum (ER) is an essential cellular compartment responsible for Ca2+ sequestration, signaling, protein translation, folding as well as transport. Several acute and chronic disease conditions impair ER function leading to ER stress. To study the impact of ER stress on synaptic transmission we applied tunicamycin (TM) or thapsigargin (TG) to hippocampal neurons, which triggered sustained elevation of key ER stress markers. We monitored evoked and spontaneous neurotransmission during 4 d of TM or TG treatment and detected only a 20% increase in paired pulse depression suggesting an increase in neurotransmitter release probability. However, the treatments did not significantly affect the number of active synapses or the size of the total recycling vesicle pool as measured by uptake and release of styryl dye FM1-43. In contrast, under the same conditions, we observed a dramatic fourfold increase in spontaneous excitatory transmission, which could be reversed by chronic treatment with the NMDA receptor blocker AP-5 or by treatment with salubrinal, a selective inhibitor of eukaryotic translation initiation factor 2 (eIF2α) dephosphorylation. Furthermore, ER stress caused NMDA receptor-dependent suppression of eukaryotic elongation factor-2 (eEF2) phosphorylation thus reversing downstream signaling mediated by spontaneous release. Together, these findings suggest that chronic ER stress augments spontaneous excitatory neurotransmission and reverses its downstream signaling in a NMDA receptor-dependent manner, which may contribute to neuronal circuitry abnormalities that precede synapse degeneration in several neurological disorders.