Loss of GluN2A‐containing NMDA receptors impairs extra‐dimensional set‐shifting

Glutamate neurotransmission via the N‐methyl‐d ‐aspartate receptor (NMDAR) is thought to mediate the synaptic plasticity underlying learning and memory formation. There is increasing evidence that deficits in NMDAR function are involved in the pathophysiology of cognitive dysfunction seen in neuropsychiatric disorders and addiction. NMDAR subunits confer different physiological properties to the receptor, interact with distinct intracellular postsynaptic scaffolding and signaling molecules, and are differentially expressed during development. Despite these known differences, the relative contribution of individual subunit composition to synaptic plasticity and learning is not fully elucidated. We have previously shown that constitutive deletion of GluN2A subunit in the mouse impairs discrimination and re‐learning phase of reversal when exemplars are complex picture stimuli, but spares acquisition and extinction of non‐discriminative visually cued instrumental response. To investigate the role of GluN2A containing NMDARs in executive control, we tested GluN2A knockout (GluN2A^KO), heterozygous (GluN2A^HET) and wild‐type (WT) littermates on an attentional set‐shifting task using species‐specific stimulus dimensions. To further explore the nature of deficits in this model, mice were tested on a visual discrimination reversal paradigm using simplified rotational stimuli. GluN2A^KO were not impaired on discrimination or reversal problems when tactile or olfactory stimuli were used, or when visual stimuli were sufficiently easy to discriminate. GluN2A^KO showed a specific and significant impairment in ventromedial prefrontal cortex‐mediated set‐shifting. Together these results support a role for GluN2A containing NMDAR in modulating executive control that can be masked by overlapping deficits in attentional processes during high task demands.     

Adenosine A2A receptors are required for glutamate mGluR5‐ and dopamine D1 receptor‐evoked ERK1/2 phosphorylation in rat hippocampus: involvement of NMDA receptor

Interaction between mGluR5 and NMDA receptors (NMDAR ) is vital for synaptic plasticity and cognition. We recently demonstrated that stimulation of mGluR5 enhances NMDAR responses in hippocampus by phosphorylating NR2B(Tyr1472) subunit, and this reaction was enabled by adenosine A_2A receptors (A_2AR) (J Neurochem, 135, 2015, 714). In this study, by using in vitro phosphorylation and western blot analysis in hippocampal slices of male Wistar rats, we show that mGluR5 stimulation or mGluR5/NMDAR s co‐stimulation synergistically activate ERK 1/2 signaling leading to c‐Fos expression. Interestingly, both reactions are under the permissive control of endogenous adenosine acting through A_2ARs. Moreover, mGluR5‐mediated ERK 1/2 phosphorylation depends on NMDAR , which however exhibits a metabotropic way of function, since no ion influx through its ion channel is required. Furthermore, our results demonstrate that mGluR5 and mGluR5/NMDAR ‐evoked ERK 1/2 activation correlates well with the mGluR5/NMDAR ‐evoked NR2B(Tyr1472) phosphorylation, since both phenomena coincide temporally, are Src dependent, and are both enabled by A_2ARs. This indicates a functional involvement of NR2B(Tyr1472) phosphorylation in the ERK 1/2 activation. Our biochemical results are supported by electrophysiological data showing that in CA 1 region of hippocampus, the theta burst stimulation (TBS)‐induced long‐term potentiation coincides temporally with an increase in ERK 1/2 activation and both phenomena are dependent on the tripartite A_2A, mGlu5, and NMDAR s. Furthermore, we show that the dopamine D1 receptors evoked ERK 1/2 activation as well as the NR2B(Tyr1472) phosphorylation are also regulated by endogenous adenosine and A_2ARs. In conclusion, our results highlight the A_2ARs as a crucial regulator not only for NMDAR responses, but also for regulating ERK 1/2 signaling and its downstream pathways, leading to gene expression, synaptic plasticity, and memory consolidation.

     

Combined brain‐derived neurotrophic factor with extinction training alleviate impaired fear extinction in an animal model of post‐traumatic stress disorder

Impaired fear memory extinction (Ext) is one of the hallmark symptoms of post‐traumatic stress disorder (PTSD). However, since the precise mechanism of impaired Ext remains unknown, effective interventions have not yet been established. Recently, hippocampal‐prefrontal brain‐derived neurotrophic factor (BDNF) activity was shown to be crucial for Ext in naïve rats. We therefore examined whether decreased hippocampal‐prefrontal BDNF activity is also involved in the Ext of rats subjected to a single prolonged stress (SPS) as a model of PTSD. BDNF levels were measured by enzyme‐linked immunosorbent assay (ELISA), and phosphorylation of TrkB was measured by immunohistochemistry in the hippocampus and medial prefrontal cortex (mPFC) of SPS rats. We also examined whether BDNF infusion into the ventral mPFC or hippocampus alleviated the impaired Ext of SPS rats in the contextual fear conditioning paradigm. SPS significantly decreased the levels of BDNF in both the hippocampus and mPFC and TrkB phosphorylation in the ventral mPFC. Infusion of BDNF 24 hours after conditioning in the infralimbic cortex (ILC), but not the prelimbic cortex (PLC) nor hippocampus, alleviated the impairment of Ext. Since amelioration of impaired Ext by BDNF infusion did not occur without extinction training, it seems the two interventions must occur consecutively to alleviate impaired Ext. Additionally, BDNF infusion markedly increased TrkB phosphorylation in the ILC of SPS rats. These findings suggest that decreased BDNF signal transduction might be involved in the impaired Ext of SPS rats, and that activation of the BDNF‐TrkB signal might be a novel therapeutic strategy for the impaired Ext by stress.     

Brain‐derived neurotrophic factor attenuates doxorubicin‐induced cardiac dysfunction through activating Akt signalling in rats

The clinical application of doxorubicin (Dox) is limited by its adverse effect of cardiotoxicity. Previous studies have suggested the cardioprotective effect of brain‐derived neurotrophic factor (BDNF). We hypothesize that BDNF could protect against Dox‐induced cardiotoxicity. Sprague Dawley rats were injected with Dox (2.5 mg/kg, 3 times/week, i.p.), in the presence or absence of recombinant BDNF (0.4 μg/kg, i.v.) for 2 weeks. H9c2 cells were treated with Dox (1 μM) and/or BDNF (400 ng/ml) for 24 hrs. Functional roles of BDNF against Dox‐induced cardiac injury were examined both in vivo and in vitro. Protein level of BDNF was reduced in Dox‐treated rat ventricles, whereas BDNF and its receptor tropomyosin‐related kinase B (TrkB) were markedly up‐regulated after BDNF administration. Brain‐derived neurotrophic factor significantly inhibited Dox‐induced cardiomyocyte apoptosis, oxidative stress and cardiac dysfunction in rats. Meanwhile, BDNF increased cell viability, inhibited apoptosis and DNA damage of Dox‐treated H9c2 cells. Investigations of the underlying mechanisms revealed that BDNF activated Akt and preserved phosphorylation of mammalian target of rapamycin and Bad without affecting p38 mitogen‐activated protein kinase and extracellular regulated protein kinase pathways. Furthermore, the beneficial effect of BDNF was abolished by BDNF scavenger TrkB‐Fc or Akt inhibitor. In conclusion, our findings reveal a potent protective role of BDNF against Dox‐induced cardiotoxicity by activating Akt signalling, which may facilitate the safe use of Dox in cancer treatment.     

Beta‐amyloid 1‐42 monomers,but not oligomers,produce PHF‐like conformation of Tau protein

The mechanistic relationship between amyloid β1‐42 (Aβ1‐42) and the alteration of Tau protein are debated. We investigated the effect of Aβ1‐42 monomers and oligomers on Tau, using mice expressing wild‐type human Tau that do not spontaneously develop Tau pathology. After intraventricular injection of Aβ1‐42, mice were sacrificed after 3 h or 4 days. The short‐lasting treatment with Aβ monomers, but not oligomers, showed a conformational PHF‐like change of Tau, together with hyperphosphorylation. The same treatment induced increase in concentration of GSK3 and MAP kinases. The inhibition of the kinases rescued the Tau changes. Aβ monomers increased the levels of total Tau, through the inhibition of proteasomal degradation. Aβ oligomers reproduced all the aforementioned alterations only after 4 days of treatment. It is known that Aβ1‐42 monomers foster synaptic activity. Our results suggest that Aβ monomers physiologically favor Tau activity and dendritic sprouting, whereas their excess causes Tau pathology. Moreover, our study indicates that anti‐Aβ therapies should be targeted to Aβ1‐42 monomers too.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.     

Differential effects of transient and sustained activation of BDNF‐TrkB signaling

Brain‐derived neurotrophic factor (BDNF) serves a pleiotropic role in the central nervous system, ranging from promoting neuronal survival and differentiation during development and synaptic modulation in the adult. An important, yet unanswered question is how BDNF could serve such diverse functions, sometimes in the same cell. At least two modes of BDNF actions have been elucidated so far based on BDNF signaling kinetics and/or the activity status of the responding neurons. Acute and gradual increases in extracellular BDNF concentrations elicit, respectively, transient and sustained activation of TrkB receptor and its downstream signaling, leading to differential molecular and cellular functions. In cultured neurons, sustained TrkB activation promotes neuronal dendritic arborization and spinogenesis, whereas transient TrkB activation facilitates dendritic growth and spine morphogenesis. In hippocampal slices, slow delivery of BDNF facilitates LTP, whereas fast application of BDNF enhances basal synaptic transmission in schaffer collateral synapses. High‐frequency stimulation of neurons converts BDNF‐induced TrkB signaling from a transient to a sustained mode. These initial insights lay the foundation for future investigation of the BDNF‐TrkB pathway, and analogous signaling pathways to gain a comprehensive understanding to enable translational research. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 647–659, 2018     

Ethanol alters trafficking and functional N-methyl-D-aspartate receptor NR2 subunit ratio via H-Ras

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity and is one of the main targets for alcohol (ethanol) in the brain. Trafficking of the NMDAR is emerging as a key regulatory mechanism that underlies channel activity and synaptic plasticity. Here we show that exposure of hippocampal neurons to ethanol increases the internalization of the NR2A but not NR2B subunit of the NMDAR via the endocytic pathway. We further observed that ethanol exposure results in NR2A endocytosis through the activation of H-Ras and the inhibition of the tyrosine kinase Src. Importantly, ethanol treatment alters functional subunit composition from NR2A/NR2B- to mainly NR2B-containing NMDARs. Our results suggest that addictive drugs such as ethanol alter NMDAR trafficking and subunit composition. This may be an important mechanism by which ethanol exerts its effects on NMDARs to produce alcohol-induced aberrant plasticity.     

Repeated cocaine enhances ventral hippocampal‐stimulated dopamine efflux in the nucleus accumbens and alters ventral hippocampal NMDA receptor subunit expression

Dopaminergic neurotransmission in the nucleus accumbens is important for various reward‐related cognitive processes including reinforcement learning. Repeated cocaine enhances hippocampal synaptic plasticity, and phasic elevations of accumbal dopamine evoked by unconditioned stimuli are dependent on impulse flow from the ventral hippocampus. Therefore, sensitized hippocampal activity may be one mechanism by which drugs of abuse enhance limbic dopaminergic activity. In this study, in vivo microdialysis in freely moving adult male Sprague–Dawley rats was used to investigate the effect of repeated cocaine on ventral hippocampus‐mediated dopaminergic transmission within the medial shell of the nucleus accumbens. Following seven daily injections of saline or cocaine (20 mg/kg, ip), unilateral infusion of N‐methyl‐d ‐aspartate (NMDA, 0.5 μg) into the ventral hippocampus transiently increased both motoric activity and ipsilateral dopamine efflux in the medial shell of the nucleus accumbens, and this effect was greater in rats that received repeated cocaine compared to controls that received repeated saline. In addition, repeated cocaine altered NMDA receptor subunit expression in the ventral hippocampus, reducing the NR2A : NR2B subunit ratio. Together, these results suggest that repeated exposure to cocaine produces maladaptive ventral hippocampal‐nucleus accumbens communication, in part through changes in glutamate receptor composition.

     

Tau inhibits PKA by nuclear proteasome‐dependent PKAR2α elevation with suppressed CREB/GluA1 phosphorylation

Intraneuronal accumulation of wild‐type tau plays a key role in Alzheimer's disease, while the mechanisms underlying tauopathy and memory impairment remain unclear. Here, we report that overexpressing full‐length wild‐type human tau (hTau) in mouse hippocampus induces learning and memory deficits with remarkably reduced levels of multiple synapse‐ and memory‐associated proteins. Overexpressing hTau inhibits the activity of protein kinase A (PKA) and decreases the phosphorylation level of cAMP‐response element binding protein (CREB), GluA1, and TrkB with reduced BDNF mRNA and protein levels both in vitro and in vivo. Simultaneously, overexpressing hTau increased PKAR2α (an inhibitory subunit of PKA) in nuclear fraction and inactivated proteasome activity. With an increased association of PKAR2α with PA28γ (a nuclear proteasome activator), the formation of PA28γ‐20S proteasome complex remarkably decreased in the nuclear fraction, followed by a reduced interaction of PKAR2α with 20S proteasome. Both downregulating PKAR2α by shRNA and upregulating proteasome by expressing PA28γ rescued hTau‐induced PKA inhibition and CREB dephosphorylation, and upregulating PKA improved hTau‐induced cognitive deficits in mice. Together, these data reveal that intracellular tau accumulation induces synapse and memory impairments by inhibiting PKA/CREB/BDNF/TrkB and PKA/GluA1 signaling, and deficit of PA28γ‐20S proteasome complex formation contributes to PKAR2α elevation and PKA inhibition.     

Inhibition of protein kinase C reduces ischemia-induced tyrosine phosphorylation of the N-methyl-d-aspartate receptor

The role of protein kinase C (PKC) in tyrosine phosphorylation of the N‐methyl‐d ‐aspartate receptor (NMDAR) following transient cerebral ischemia was investigated. Transient (15 min) cerebral ischemia was produced in adult rats by four‐vessel occlusion and animals allowed to recover for 15 or 45 min. Following ischemia, tyrosine phosphorylation of NR2A and NR2B and activated Src‐family kinases (SFKs) and Pyk2 were increased in post‐synaptic densities (PSDs). Phosphorylation of NR2B on Y1472 by PSDs isolated from post‐ischemic forebrains was inhibited by the SFK specific inhibitor PP2, and by the PKC inhibitors GF109203X (GF), Gö6976 and calphostin C. Intravenous injection of GF immediately following the ischemic challenge resulted in decreased phosphorylation of NR1 on PKC phosphorylation sites and reduced ischemia‐induced increases in tyrosine phosphorylation of NR2A and NR2B without affecting the increase in total tyrosine phosphorylation of hippocampal proteins. Ischemia‐induced increases in activated Pyk2 and SFKs in PSDs, but not the translocation of PKC, Pyk2 or Src to the PSD, were also inhibited by GF. The inactive homologue of GF, bisindolylmaleimide V, had no effect on these parameters. The results are consistent with a role for PKC in the ischemia‐induced increase in tyrosine phosphorylation of the NMDAR, via a pathway involving Pyk2 and Src‐family kinases.     

SORLA-Mediated Trafficking of TrkB Enhances the Response of Neurons to BDNF

Stimulation of neurons with brain-derived neurotrophic factor (BDNF) results in robust induction of SORLA, an intracellular sorting receptor of the VPS10P domain receptor gene family. However, the relevance of SORLA for BDNF-induced neuronal responses has not previously been investigated. We now demonstrate that SORLA is a sorting factor for the tropomyosin-related kinase receptor B (TrkB) that facilitates trafficking of this BDNF receptor between synaptic plasma membranes, post-synaptic densities, and cell soma, a step critical for neuronal signal transduction. Loss of SORLA expression results in impaired neuritic transport of TrkB and in blunted response to BDNF in primary neurons; and it aggravates neuromotoric deficits caused by low BDNF activity in a mouse model of Huntington’s disease. Thus, our studies revealed a key role for SORLA in mediating BDNF trophic signaling by regulating the intracellular location of TrkB.     

Diazepam improves aspects of social behaviour and neuron activation in NMDA receptor‐deficient mice

NR1 knockdown (NR1KD) mice are genetically modified to express low levels of the NR1 subunit of N‐methyl‐d ‐aspartate (NMDA) receptors, and show deficits in affiliative social behaviour. In this study, we determined which brain regions were selectively activated in response to social stimulation and asked whether differences in neuronal activation could be observed in mice with reduced sociability. Furthermore, we aimed to determine whether brain activation patterns correlated with the amelioration of social deficits through pharmacological intervention. The cingulate cortex, lateral septal nuclei, hypothalamus, thalamus and amygdala showed an increase in c‐Fos immunoreactivity that was selective for exposure to social stimuli. NR1KD mice displayed a reduction in social behaviour and a reduction in c‐Fos immunoreactivity in the cingulate cortex and septal nuclei. Acute clozapine did not significantly alter sociability; however, diazepam treatment did increase sociability and neuronal activation in the lateral septal region. This study has identified the lateral septal region as a neural substrate of social behaviour and the GABA system as a potential therapeutic target for social dysfunction.     

A steroid modulatory domain in NR2A collaborates with NR1 exon-5 to control NMDAR modulation by pregnenolone sulfate and protons

NMDA receptor (NMDAR)-mediated excitatory synaptic transmission plays a critical role in synaptic plasticity and memory formation, whereas its dysfunction may underlie neuropsychiatric and neurodegenerative diseases. The neuroactive steroid pregnenolone sulfate (PS) acts as a cognitive enhancer in impaired animals, augments LTP in hippocampal slices by enhancing NMDAR activity, and may participate in the reduction of schizophrenia's negative symptoms by systemic pregnenolone. We report that the effects of PS on NMDAR function are diverse, varying with subunit composition and NR1 splice variant. While PS potentiates NR1-1a/NR2B receptors through a critical steroid modulatory domain in NR2B that also modulates tonic proton inhibition, potentiation of the NMDA response is not dependent upon relief of such inhibition, a finding that distinguishes it from spermine. In contrast, the presence of an NR2A subunit confers enhanced PS-potentiation at reduced pH, suggesting that it may indeed act like spermine does at NR2B-containing receptors. Additional tuning of the NMDAR response by PS comes via the N-terminal exon-5 splicing insert of NR1-1b, which regulates the magnitude of proton-dependent PS potentiation. For NR2C- and NR2D-containing receptors, negative modulation at NR2C receptors is pH-independent (like NR2B) while negative modulation at NR2D receptors is pH-dependent (like NR2A). Taken together, PS displays a rich modulatory repertoire that takes advantage of the structural diversity of NMDARs in the CNS. The differential pH sensitivity of NMDAR isoforms to PS modulation may be especially important given the emerging role of proton sensors to both learning and memory, as well as brain injury.     

Altered NMDA receptor trafficking contributes to sleep deprivation-induced hippocampal synaptic and cognitive impairments

Recent evidence indicates that continuous wakefulness (sleep deprivation, SD) causes impairments in behavioral performance and hippocampal long-term potentiation (LTP) in animals. However, the mechanisms by which SD impairs long-term synaptic plasticity and cognitive function are not clear. Here, we report that 24-h SD in mice results in impaired hippocampus-dependent contextual memory and LTP and, unexpectedly, in reductions of the surface expression of NMDA receptor (NMDAR) subunit NR1 and NMDAR-mediated excitatory post-synaptic currents at hippocampal perforant path-dentate granule cell synapses. The results suggest that the reduction of functional NMDAR in hippocampal neurons may underlie the SD-induced deficits in hippocampus-dependent contextual memory and long-term synaptic plasticity.