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Enhanced tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate receptor in long-term potentiation.
Abstract
Both serine/threonine and tyrosine phosphorylation of receptor proteins have been implicated in the process of long-term potentiation (LTP), but there has been no direct demonstration of a change in receptor phosphorylation after LTP induction. We show that, after induction of LTP in the dentate gyrus of anesthetized adult rats, there is an increase in the tyrosine phosphorylation of the 2B subunit of the N-methyl-D-aspartate (NMDA) receptor (NR2B), as well as several other unidentified proteins. Tyrosine phosphorylation of NR2B was measured in two ways: binding of antiphosphotyrosine antibodies (PY20) to glycoprotein(s) of 180 kDa (GP180) purified on Con A-Sepharose and binding of anti-NR2B antibodies to tyrosine-phosphorylated proteins purified on PY20-agarose. Three hours after LTP induction, anti-NR2B binding to tyrosine phosphorylated proteins, expressed as a ratio of tetanized to control dentate (Tet/Con), was 2.21 +/- 0.50 and PY20 binding to GP180 was 1.68 +/- 0.16. This increase in the number of tyrosine phosphorylated NR2B subunits occurred without a change in the total number of NR2B subunits. When the induction of LTP was blocked by pretreatment of the animal with the NMDA receptor antagonist MK801, the increase in PY20 binding to GP180 was also blocked (Tet/Con = 1.09 +/- 0.26). The increased PY20 binding to GP180 was also apparent 15 min after LTP induction (Tet/Con = 1.41 +/- 0.16) but not detectable 5 min after LTP induction (Tet/Con = 1.01 +/- 0.19). These results suggest that tyrosine phosphorylation of the NMDA receptor contributes to the maintenance of LTP.
... Moreover, 4 months of treatment with FA prevented the observed loss of p-Y1472 GluN2B in APP/ PS1 mice. It has been demonstrated that GluN2B is the most prominent tyrosine-phosphorylated protein within PSDs [103], and its phosphorylation is upregulated during LTP in the CA1 hippocampus [104]. The synaptic expression of NMDARs is significantly regulated by their phosphorylation [105], and the activity-dependent subunit-specific phosphorylation of NMDARs has a significant impact on their synaptic localization and function [106,107]. ...
... PSD-95 is a scaffolding protein, the major component of PSD, that functions as a PSD organizer and is central to glutamatergic synaptic signaling [117]. PSD-95 is reported to stabilize the surface expression of GluN2B-containing NMDARs by its direct interaction with the GluN2B PDZ ligand and by triggering STEP ubiquitination and degradation [5,9], resulting in increased GluN2B phosphorylation during LTP [104]. PSD-95 also interacts with cysteine-rich PDZ-binding protein and forms a linkage with the neuronal cytoskeleton to regulate dendritic arborization and spine number [118]. ...
The burden of Alzheimer's disease, the most prevalent neurodegenerative disease, is increasing exponentially due to the increase in the elderly population worldwide. Synaptic plasticity is the basis of learning and memory, but it is impaired in AD. Uncovering the disease's underlying molecular pathogenic mechanisms involving synaptic plasticity could lead to the identification of targets for better disease management. Using primary neurons treated with Aβ and APP/PS1 animal models, we evaluated the effect of the phenolic compound ferulic acid (FA) on synaptic dysregulations. Aβ led to synaptic plasticity and cognitive impairments by increasing STEP activity and decreasing the phosphorylation of the GluN2B subunit of NMDA receptors, as well as decreasing other synaptic proteins, including PSD-95 and synapsin1. Interestingly, FA attenuated the Aβ-upregulated intracellular calcium and thus resulted in a decrease in PP2B-induced activation of DARPP-32, inhibiting PP1. This cascade event maintained STEP in its inactive state, thereby preventing the loss of GluN2B phosphorylation. This was accompanied by an increase in PSD-95 and synapsin1, improved LTP, and a decreased Aβ load, together leading to improved behavioral and cognitive functions in APP/PS1 mice treated with FA. This study provides insight into the potential use of FA as a therapeutic strategy in AD.
... To examine potential mechanisms underlying the memory impairments induced by isoflurane and HFD/STZ treatment, we measured the effects of the first-line antidiabetic drug metformin on cognitive performance, IR, and phosphorylation of the NMDA receptor, which is essential for many forms of synaptic plasticity underlying hippocampus-dependent learning and memory (Rostas et al., 1996;Petrone et al., 2003). Metformin has been shown to cross the blood-brain barrier rapidly and effectively ameliorate IR (Giannarelli et al., 2003;Łabuzek et al., 2010). ...
... Consistent with these clinical findings, isoflurane upregulated the expression of p-tau in hippocampus, while metformin decreased the expression of hippocampal p-tau. As a cytoskeleton-associated protein, tau plays an important role in the postsynaptic targeting of the Scr kinase Fyn to NMDA receptors, where it phosphorylates the NR2B subunit at Tyr-1472 and promotes NMDAR-dependent synaptic plasticity (Rostas et al., 1996;Petrone et al., 2003;Ittner et al., 2010). Moreover, hyperphosphorylated tau has been shown to reduce Fyn binding (Reynolds et al., 2008). ...
General anesthetics can induce cognitive impairments and increase the risk of Alzheimer’s disease (AD). However, the underlying mechanisms are still unknown. Our previous studies shown that long-term isoflurane exposure induced peripheral and central insulin resistance (IR) in adult mice and aggravated IR in type 2 diabetes mellitus (T2DM) mice. Clinical and preclinical studies revealed an association between impaired insulin signaling and tau pathology in AD and other tauopathies. We investigated if alleviation of hippocampal IR by the antidiabetic agent metformin could reduce tau hyperphosphorylation and cognitive decline induced by isoflurane in mice. The effects of prolonged (6 h) isoflurane anesthesia on hippocampal IR, hippocampal tau hyperphosphorylation, and hippocampus-dependent cognitive function were evaluated in wild type (WT) adult mice and the high-fat diet plus streptozotocin (HFD/STZ) mouse model of T2DM. Here we shown that isoflurane and HFD/STZ dramatically and synergistically induced hippocampal IR and fear memory impairment. Metformin pretreatment strongly ameliorated hippocampal IR and cognitive dysfunction caused by isoflurane in WT mice, but was less effective in T2DM mice. Isoflurane also induced hippocampal tau hyperphosphorylation and metformin reversed this effect. In addition, isoflurane significantly increased blood glucose levels in both adult and T2DM mice, and metformin reversed this effect as well. Administration of 25% glucose to metformin-pretreated mice induced hyperglycemia, but surprisingly did not reverse the benefits of metformin on hippocampal insulin signaling and fear memory following isoflurane anesthesia. Our findings show hippocampal IR and tau hyperphosphorylation contribute to acute isoflurane-induced cognitive dysfunction. Brief metformin treatment can mitigate these effects through a mechanism independent of glycemic control. Future studies are needed to investigate whether long-term metformin treatment can also prevent T2DM-induced hippocampal IR and cognitive decline.
... Furthermore, it has been demonstrated that NMDARdependent long-term potentiation (LTP) in the hippocampus can be blocked by the inhibition of SFKs and accompanied by both the Src-mediated upregulation of NMDAR conductance and an increased phosphorylation of GluN2B subunits on tyrosine residues (Rostas et al. 1996;Lu et al. 1998). Another finding suggests that SFKs, NMDAR stimulation, and possible SFKs-mediated GluN2B-containing NMDAR activation in the hippocampus are necessary for memory processes associated with contextual cocaine-seeking behavior (Xie et al. 2013). ...
Memories are stored into long-term representations through a process that depends on protein synthesis. However, a consolidated memory is not static and inflexible and can be reactivated under certain circumstances, the retrieval is able to reactivate memories and destabilize them engaging a process of restabilization known as reconsolidation. Although the molecular mechanisms that mediate fear memory reconsolidation are not entirely known, so here we investigated the molecular mechanisms in the hippocampus involved in contextual fear conditioning memory (CFC) reconsolidation in male Wistar rats. We demonstrated that the blockade of Src family kinases (SFKs), GluN2B-containing NMDA receptors and TrkB receptors (TrkBR) in the CA1 region of the hippocampus immediately after the reactivation session impaired contextual fear memory reconsolidation. These impairments were blocked by the neurotrophin BDNF and the NMDAR agonist, D-Serine. Considering that the study of the link between synaptic proteins is crucial for understanding memory processes, targeting the reconsolidation process may provide new ways of disrupting maladaptive memories, such as those seen in post-traumatic stress disorder. Here we provide new insights into the cellular mechanisms involved in contextual fear memory reconsolidation, demonstrating that SFKs, GluN2B-containing NMDAR, and TrkBR are necessary for the reconsolidation process. Our findings suggest a link between BDNF and SFKs and GluN2B-containing NMDAR as well as a link between NMDAR and SFKs and TrkBR in fear memory reconsolidation. These preliminary pharmacological findings provide new evidence of the mechanisms involved in the reconsolidation of fear memory and have the potential to contribute to the development of treatments for psychiatric disorders involving maladaptive memories.
... Both NR2A and NR2B subunits are known to be phosphorylated at C-terminal tyrosine residues by intracellular tyrosine kinases, that potentiate ion channel current through recombinant channels of NR1-NR2A and/or NR1-NR2B, thus relieving the basal zinc inhibition of NMDA receptors (Zheng et al., 1998). Interesting observations have demonstrated that induction of LTP produces an activation of SRC within 1-5min (Lu et al., 1998) with the resultant tyrosine-residue phosphorylation of NR2B (Rostas et al., 1996;Rosenblum et al., 1996). SRC infusion (as LTP does) produces a significant enhancement of AMPA-receptor-mediated current that is dependent on the activation of NMDA-receptor channel and Ca 2+ influx and, conversely, application of SRC-specific inhibitors prior to HFS, prevents LTP induction (Lu et al., 1998). ...
Extensive cellular and behavioral studies have led to the postulation that memories are encoded by changes in synaptic strength between neurons, as demonstrated by the correlation between long-term changes in animal behavior and long-term changes in neuronal connections underlying a specific behavior in invertebrate animals, or even in vertebrate animals, where cellular models of synaptic plasticity using genetic approaches, such as Long-Term Potentiation (LTP) and Long-Term Depression (LTD), have been shown to depend on long-term changes in synaptic activity implicated in behavioral learning and memory. Long-term memory (LTM) is crucial for animal's survival and thus represents a mechanism that underlies fundamental neurobiological events in the nervous system of vertebrate and non-vertebrate species including the human. Long-term changes in synaptic connectivity as well as long-term behavioral changes (both activities that underlie several of the properties of LTM and are used as a parameter to explain the long-lasting enhancement of neuronal function after a stimulus) have been demonstrated to rely on signals that initially occur in the cell body. LTP is a form of synaptic plasticity widely accepted as a cellular model for stabilization of synapses in neurobiological phenomena such as development and learning and memory. Much of the experimental work concerning LTP in learning has been focused on the NMDA receptor dependent forms of LTP. But several questions have arisen regarding if LTP equals memory. If LTP has a real role in memory, a more appropriate hypothesis should be stated by postulating that activity-dependent synaptic plasticity and multiple forms of memory known to exist, share a common core; that is, the synaptic plasticity and memory hypothesis states that activity dependent synaptic plasticity is induced at appropriate synapses during memory formation. Synaptic plasticity is a physiological phenomenon that induces specific patterns of neural activity sustained by chemical and molecular mechanisms, that gives rise to changes in synaptic efficacy and neural excitability that long outlast the events that trigger them. Based on the various properties of synaptic plasticity discovered, LTP may be proposed as a suitable neuronal mechanism for the development of several memory systems, including initial encoding and storage of memory traces and initial phases of trace consolidation over time. Such memory processing made up by LTP or LTD most probably occur as a network specific process, making LTP a universal mechanism for encoding and storage of memory traces and, what gets encoded, is part of a network property rather than mechanisms working at individual synapses. For example, the type of information processed at the hippocampus is quite different from the information processed by the amygdala, and such information should remain if the mechanisms of plasticity operating in each brain area are conserved. Decades of research have demonstrated that LTP in the hippocampus is induced by synaptic activity and that cytoplasmic membrane-bound molecule(s) are required to transduce extracellular signals mediated by receptor-activation into activation of intracellular signaling processes. Most of these processes depend on intracellular calcium activity, and thereby on calcium-dependent mechanisms that are recruited for LTP induction and expression. For instance, NMDA receptors have been shown to be essential for initiation of LTP, but the expression of this phenomenon is brought about primarily by AMPA receptors. Induction of LTP in CA1 hippocampal region has been shown to depend on increases of intracellular calcium and activation of specific calcium-dependent molecules such as the calcium/calmodulin-dependent protein kinase (CaMKII), whose cell expression is confined predominantly at postsynaptic densities. Moreover, long-term expression of LTP requires protein synthesis, where transient signals will be linked to activation of specific genes that ultimately will determine growth and remodeling of potential active synapses. Different types of synapses may express and use a different set of molecules mediating activation of intracellular signaling pathways to initiate and maintain synaptic
... Although crucial for many normal functions, in PD, hyperphosphorylation and the resulting overactivation of NMDA receptors is a known driver of glutamate excitotoxicity (Iovino et al., 2020;Truong et al., 2009). Additionally, increased levels of NR2B subunit phosphorylation have been observed not only in the striatum (Dunah et al., 2000;Oh et al., 1998), but also in the hippocampus of 6-OHDA lesioned rats (Rostas et al., 1996), which may help to account for the impairments in recognition memory seen in our 6-OHDA model. Whilst Fyn-mediated phosphorylation is vital for normal hippocampal LTP, there appears to be an optimal activity range and excessive activation or inhibition may impact performance (Parsons et al., 2007). ...
Fyn kinase has recently been established as a major upstream regulator of neuroinflammation in PD. This study aimed to determine if inhibition of Fyn kinase could lead to reduced neuroinflammation and improvements in motor and non-motor impairments in an early-stage model of PD. An experimental model of PD was produced using intra-striatal injection (4µl) of the neurotoxin 6-OHDA (5µg/µl). Sprague Dawley rats (n=42) were given either vehicle, 6mg/kg or 12mg/kg of Fyn kinase inhibitor (AZD0530) daily for 32 days via oral gavage and tested on a battery of tasks assessing motor, cognitive and neuropsychiatric outcomes. AZD 0530 administration led to improvement in volitional locomotion and recognition memory, as well as a reduction in depressive-like behaviour. Pathologically, an inflammatory response was observed; however, there were no significant differences in markers of neuroinflammation between treatment groups. Taken together, results indicate a potential therapeutic benefit for use of Fyn kinase inhibition to treat non-motor symptoms of PD, although mechanisms remain to be elucidated.
HIGHLIGHTS
Fyn kinase has recently been proposed as a major upstream regulator of microglial activation in Parkinson’s disease (PD).
This study was the first to evaluate the effects of Fyn kinase inhibition in a rodent model of PD.
Fyn kinase inhibition using the Fyn kinase inhibitor AZD 0530 was capable of improving volitional locomotion and recognition memory and reducing depressive-like behaviour in a rodent model of PD.
Interestingly, while increases in microglial activation were observed in this rodent model of PD, AZD 0530 did not significantly reduce this activation.
This suggests that the behavioural improvements associated with Fyn kinase inhibition may occur independently of neuroinflammation and may be attributable to other brain mechanisms, including actions on NMDA or 5-HT 6 receptors.
... Tyrosine phosphorylation of the GluN2B subunit increases the activity of NMDAR and is associated with the induction of LTP by the calcium/calmodulin-dependent kinase CamkII (Nicoll, 2017;Rosenblum et al., 1996;Rostas et al., 1996). However, the reduced tyrosine phosphorylation of GluN2B-Y 1472 we observed in hAPP-20 mice did not seem to affect post-synaptic NMDAR-mediated eEPSC amplitudes or the NMDAR/AMPAR ratio, consistent with a previous study of GluN2B-Y 1472 F knock-in mice expressing a non-phosphorylatable GluN2B subunit (Nakazawa et al., 2006). ...
Mutations in the beta-amyloid protein (APP) cause familial Alzheimer's disease. In hAPP-J20 mice expressing mutant APP, pharmacological inhibition or genetic ablation of the tyrosine phosphatase PTP1B prevents CA3 hippocampus neuron loss and cognitive decline. However, how targeting PTP1B affects the cellular mechanisms underlying these cognitive deficits remains unknown. Changes in synaptic strength at the hippocampus can affect information processing for learning and memory. While prior studies have focused on post-synaptic mechanisms to account for synaptic deficits in Alzheimer's disease models, presynaptic mechanisms may also be affected. Here, using whole cell patch-clamp recording, coefficient of variation (CV) analysis suggested a profound presynaptic deficit in long-term potentiation (LTP) of CA3:CA1 synapses in hAPP-J20 mice. While the membrane-impermeable ionotropic NMDA receptor (NMDAR) blocker norketamine in the post-synaptic recording electrode had no effect on LTP, additional bath application of the ionotropic NMDAR blockers MK801 could replicate the deficit in LTP in wild type mice. In contrast to LTP, the paired-pulse ratio and short-term facilitation (STF) were aberrantly increased in hAPP-J20 mice. These synaptic deficits in hAPP-J20 mice were associated with reduced phosphorylation of NMDAR GluN2B and the synaptic vesicle recycling protein NSF (N-ethylmaleimide sensitive factor). Phosphorylation of both proteins, together with synaptic plasticity and cognitive function, were restored by PTP1B ablation or inhibition by the PTP1B-selective inhibitor Trodusquemine. Taken together, our results indicate that PTP1B impairs presynaptic NMDAR-mediated synaptic plasticity required for spatial learning in a mouse model of Alzheimer's disease. Since Trodusquemine has undergone phase 1/2 clinical trials to treat obesity, it could be repurposed to treat Alzheimer's disease.
Lipid membrane nanodomains or lipid rafts are 10–200 nm diameter size cholesterol- and sphingolipid-enriched domains of the plasma membrane, gathering many proteins with different roles. Isolation and characterization of plasma membrane proteins by differential centrifugation and proteomic studies have revealed a remarkable diversity of proteins in these domains. The limited size of the lipid membrane nanodomain challenges the simple possibility that all of them can coexist within the same lipid membrane domain. As caveolin-1, flotillin isoforms and gangliosides are currently used as neuronal lipid membrane nanodomain markers, we first analyzed the structural features of these components forming nanodomains at the plasma membrane since they are relevant for building supramolecular complexes constituted by these molecular signatures. Among the proteins associated with neuronal lipid membrane nanodomains, there are a large number of proteins that play major roles in calcium signaling, such as ionotropic and metabotropic receptors for neurotransmitters, calcium channels, and calcium pumps. This review highlights a large variation between the calcium signaling proteins that have been reported to be associated with isolated caveolin-1 and flotillin-lipid membrane nanodomains. Since these calcium signaling proteins are scattered in different locations of the neuronal plasma membrane, i.e., in presynapses, postsynapses, axonal or dendritic trees, or in the neuronal soma, our analysis suggests that different lipid membrane-domain subtypes should exist in neurons. Furthermore, we conclude that classification of lipid membrane domains by their content in calcium signaling proteins sheds light on the roles of these domains for neuronal activities that are dependent upon the intracellular calcium concentration. Some examples described in this review include the synaptic and metabolic activity, secretion of neurotransmitters and neuromodulators, neuronal excitability (long-term potentiation and long-term depression), axonal and dendritic growth but also neuronal cell survival and death.
N-Methyl-d-aspartate (NMDA) receptor hyperfunction plays a key role in the pathological processes of depression and neurodegenerative diseases, whereas NMDA receptor hypofunction is implicated in schizophrenia. Considerable efforts have been made to target NMDA receptor function for the therapeutic intervention in those brain disorders. In this mini-review, we first discuss ion flux-dependent NMDA receptor signaling and ion flux-independent NMDA receptor signaling that result from structural rearrangement upon binding of endogenous agonists. Then, we review current strategies for exploring druggable targets of the NMDA receptor signaling and promising future directions, which are poised to result in new therapeutic agents for several brain disorders.
Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.
Although stroke is very often the cause of death worldwide, the burden of ischemic and hemorrhagic stroke varies between regions and over time regarding differences in prognosis, prevalence of risk factors, and treatment strategies. Excitotoxicity, oxidative stress, dysfunction of the blood-brain barrier, neuroinflammation, and lysosomal membrane permeabilization, sequentially lead to the progressive death of neurons. In this process, protein kinases-related checkpoints tightly regulate N-methyl-D-aspartate (NMDA) receptor signaling pathways. One of the major hallmarks of cerebral ischemia is excitotoxicity, characterized by overactivation of glutamate receptors leading to intracellular Ca²⁺ overload and ultimately neuronal death. Thus, reduced expression of postsynaptic density-95 protein and increased protein S-nitrosylation in neurons is responsible for neuronal vulnerability in cerebral ischemia. In this chapter death-associated protein kinases, cyclin-dependent kinase 5, endoplasmic reticulum stress-induced protein kinases, hyperhomocysteinemia-related NMDA receptor overactivation, ephrin-B-dependent amplification of NMDA-evoked neuronal excitotoxicity and lysosomocentric hypothesis have been discussed.
Mice with mutations in four nonreceptor tyrosine kinase genes, fyn, src, yes, and abl, were used to study the role of these
kinases in long-term potentiation (LTP) and in the relation of LTP to spatial learning and memory. All four kinases were expressed
in the hippocampus. Mutations in src, yes, and abl did not interfere with either the induction or the maintenance of LTP.
However, in fyn mutants, LTP was blunted even though synaptic transmission and two short-term forms of synaptic plasticity,
paired-pulse facilitation and post-tetanic potentiation, were normal. In parallel with the blunting of LTP, fyn mutants showed
impaired spatial learning, consistent with a functional link between LTP and learning. Although fyn is expressed at mature
synapses, its lack of expression during development resulted in an increased number of granule cells in the dentate gyrus
and of pyramidal cells in the CA3 region. Thus, a common tyrosine kinase pathway may regulate the growth of neurons in the
developing hippocampus and the strength of synaptic plasticity in the mature hippocampus.
As a first step in a program to use genetically altered mice in the study of memory mechanisms, mutant mice were produced that do not express the alpha-calcium-calmodulin-dependent kinase II (alpha-CaMKII). The alpha-CaMKII is highly enriched in postsynaptic densities of hippocampus and neocortex and may be involved in the regulation of long-term potentiation (LTP). Such mutant mice exhibited mostly normal behaviors and presented no obvious neuroanatomical defects. Whole cell recordings reveal that postsynaptic mechanisms, including N-methyl-D-aspartate (NMDA) receptor function, are intact. Despite normal postsynaptic mechanisms, these mice are deficient in their ability to produce LTP and are therefore a suitable model for studying the relation between LTP and learning processes.
N-METHYL-D-aspartate (NMDA) glutamate receptor properties are subject to a fine tuning by several regulatory mechanisms including phosphorylation of the receptor subunits. Here we show that two of these subunits, NR2B and NR2A, are phosphorylated on tyrosine residues in vivo, in rat striatum, where NR2B is by far the most prominent tyrosine phosphorylated protein. Two weeks after unilateral lesioning of nigrostriatal dopaminergic neurones with 6-hydroxydopamine, tyrosine phosphorylation of NR2B was increased by similar to 20% in the ipsilateral striatum. The total amount of NR2B protein was unaltered. Thus, increased tyrosine phosphorylation of NR2B may account for some of the consequences of dopamine deprivation on corticostriatal transmission and may play a role in some forms of synaptic plasticity.
: Postsynaptic densities (PSDs) were prepared by the aqueous two-phase extraction of synaptic membranes in the presence of n-octyl glucoside. Incubation of postsynaptic densities with [γ-32P]ATP resulted in the incorporation of 32P into a range of proteins. Isolation of glycoproteins from 32P-labelled PSDs by affinity chromatography on concanavalin A-agarose identified the postsynaptic glycoprotein of apparent M, 180,000 (gp180) as a substrate for endogenous protein kinase(s). When the phosphorylation reaction was performed in the presence of Ca2+ and calmodulin, there was an overall 13- fold increase in the phosphorylation of PSD proteins. The largest effects of calmodulin were associated with two proteins of molecular weights 51,000 and 60,000, which showed average calmodulin-dependent increases in phosphorylation of 68-fold. The phosphorylation of gp180 was increased 7.5-fold in the presence of calmodulin. Fifty percent of maximum phosphorylation of proteins and glycoproteins occurred with a free Ca2+ concentration of 0.3 × 10−6M. The amounts 12.6 μg/ml and 9.1 μg/ml of calmodulin were required for 50% of maximum phosphorylation of proteins and glycoproteins, respectively. Peptide mapping experiments identified three major phosphorylation sites in gp180. The phosphorylation of all three sites was increased in the presence of calmodulin. Phosphoamino acid analysis of gp180 revealed that [32P]phosphoserine and [32P]phosphothreonine were both produced during the phosphorylation reaction, with phosphoserine being the predominant product. The phosphorylation of both amino acids was increased in the presence of calmodulin. [32P]phosphotyrosine was also identified as a product of the phosphorylation of gp180.
The N-methyl d-aspartate (NMDA) receptor subtype of glutamate-gated ion channels possesses high calcium permeability and unique voltage-dependent
sensitivity to magnesium and is modulated by glycine. Molecular cloning identified three complementary DNA species of rat
brain, encoding NMDA receptor subunits NMDAR2A (NR2A), NR2B, and NR2C, which are 55 to 70% ientical in sequence. These are
structurally related, with less than 20% sequence identity, to other excitatory amino acid receptor subunits, including the
NMDA receptor subunit NMDAR1 (NR1). Upon expression in cultured cells, the new subunits yielded prominent, typical glutamate-and
NMDA-activated currents only when they were in heteromeric configurations with NR1. NR1-NR2A and NR1-NR2C channels differed
in gating behavior and magnesium sensitivity. Such heteromeric NMDA receptor subtypes may exist in neurons, since NR1 messenger
RNA is synthesized throughout the mature rat brain, while NR2 messenger RNA show a differential distribution.
THE major postsynaptic density (PSD) protein1,2 at glutaminergic synapses is calcium/calmodulin-dependent protein kinase II (CaM-K II), but its function in the PSD is not known. We have examined glutamate receptors (GluRs) as substrates for CaM-K II because (1) they are colocalized in the PSD3, (2) cloned GluRs4–7 contain consensus phosphorylation sites for protein kinases including CaM-K II, and (3) several GluRs are regulated by other protein kinases8–12. Regulation of GluRs, which are involved in excitatory synaptic transmission and in mechanisms of learning and memory13, by CaM-K II is of interest because of the postulated role of CaM-K II in synaptic plasticity14,15 and its known involvement in induction of long-term potentiation16. Furthermore, mice lacking the major neural isoform of CaM-K II exhibit deficits in models of learning and memory that require hippocampal input17,18. We report here that CaM-K II phosphorylates GluR in several in vitro systems, including the PSD, and that activated CaM-K II enhances kainate-induced ion current three- to fourfold in cultured hippocampal neurons. These results are consistent with a role for PSD CaM-K II in strengthening postsynaptic GluR responses in synaptic plasticity.
The glutamate receptors mediate excitatory neurotransmission in the brain and are important in memory acquisition, learning,
and some neurodegenerative disorders. This receptor family is classified in three groups: the N-methyl-D-aspartate (NMDA),
alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-kainate, and metabotropic receptors. Recent molecular studies
have shown that many receptor subtypes exist in all three groups of the receptors and exhibit heterogeneity in function and
expression patterns. This article reviews the molecular and functional diversity of the glutamate receptors and discusses
their implications for integrative brain function.
Previous studies on the effects of protein kinase C (PKC) inhibitors intracellularly introduced into the postsynaptic neuron on long-term potentiation (LTP) in the hippocampal CA1 region showed that given before the tetanic stimulation they only blocked the development of the maintenance phase of LTP and that given after the tetanus they did not affect the continued maintenance of established LTP. We now report different results in such experiments obtained by looking into the dose-effect relationship of the inhibitors given to the postsynaptic neuron and making use of a synergistic effect of two inhibitors given together. We used the following three PKC inhibitors: polymyxin B (PMB), PKC-(19-31), and H7. With the intracellular delivery of the inhibitor(s) beginning 30 min before the tetanus, PMB in adequate dosage or a combination of PMB and PKC-(19-31), each at a low dosage, could block the development of LTP completely including its initial induction phase. With the delivery beginning at the time of the tetanus, PKC-(19-31) or H7 slowly caused the established LTP to decline to the baseline; this decline was greatly accelerated when PMB and PKC-(19-31) or PMB and H7 were given together. PMB and PKC-(19-31) given together 75-90 min or even 3 h after the tetanus caused a decline of the maintained LTP similar to the decline observed when both inhibitors were given at the time of the tetanus. These results show that postsynaptic PKC is essentially involved in both the initial induction and the subsequent maintenance of LTP, contrary to current views on the subject.
Long-term potentiation (LTP) in the hippocampus is thought to contribute to memory formation. In the Ca1 region, LTP requires the NMDA (N-methyl-D-aspartate) receptor-dependent influx of Ca2+ and activation of serine and threonine protein kinases. Because of the high amount of protein tyrosine kinases in hippocampus and cerebellum, two regions implicated in learning and memory, we examined the possible additional requirement of tyrosine kinase activity in LTP. We first examined the specificity in brain of five inhibitors of tyrosine kinase and found that two of them, lavendustin A and genistein, showed substantially greater specificity for tyrosine kinase from hippocampus than for three serine-threonine kinases: protein kinase A, protein kinase C, and Ca2+/calmodulin kinase II. Lavendustin A and genistein selectively blocked the induction of LTP when applied in the bath or injected into the postsynaptic cell. By contrast, the inhibitors had no effect on the established LTP, on normal synaptic transmission, or on the neurotransmitter actions attributable to the actions of protein kinase A or protein kinase C. These data suggest that tyrosine kinase activity could be required postsynaptically for long-term synaptic plasticity in the hippocampus. As Ca2+ calmodulin kinase II or protein kinase C seem also to be required, the tyrosine kinases could participate postsynaptically in a kinase network together with serine and threonine kinases.
Immediate early genes (IEGs) are a class of genes that show rapid and transient but protein synthesis-independent increases in expression to extracellular signals such as growth factors and neurotransmitters. Many IEGs code for transcription factors that have been suggested to govern the growth and differentiation of many cell types by regulating the expression of other genes. IEGs are expressed in adult neurons both constitutively and in response to afferent activity, and it has been suggested that during learning, IEGs may play a role in the signal cascade, resulting in the expression of genes critical for the consolidation of long-term memory. Long-term potentiation (LTP) is a persistent, activity-dependent form of synaptic plasticity that stands as a good candidate for the mechanism of associative memory. A number of IEGs coding for transcription factors have been shown to transiently increase transcription in the dentate gyrus of rats following LTP-inducing afferent stimulation. These include zif/268 (also termed NGFI-A, Krox-24, TIS-8, and egr-l), c-fos-related genes, c-jun, junB, and junD. Of these, zif/268 appears to be the most specifically related to LTP since it is evoked under virtually all LTP-inducing situations and shows a remarkably high correlation with the duration of LTP. There are a number of outstanding questions regarding the role of zif/268 and other IEGs in LTP, including which second messenger systems are important for activating them, which "late effector" genes are regulated by them, and the exact role these genes play, if any, in the stabilization and maintenance of LTP.