Plasticity in the spinal dorsal horn may contribute to the development of pain following peripheral nerve injury. Shank proteins are a constituent family of the post-synaptic density (PSD), and they may play a role in synaptic plasticity through activity-dependent synaptic remodeling and growth. In this study we examined the early consequences of the loose ligation of the sciatic nerve on Shank1 protein and message levels in the PSD of spinal dorsal horn neurons.
Four hours after sciatic ligation, the protein levels of Shank1 increased in the ipsilateral PSD of ligated animals. In contrast, no changes were detected in the contralateral PSD of these ligated animals, or either the ipsilateral or contralateral PSD of sham-operated animals. Shank1 was linked to the PSD marker protein PSD-95 and the NR2B subunit of NMDA receptors. The ligated animals also exhibited two early signs of pain behavior, a shift in weight bearing and thermal hyperalgesia. There was no overall change in Shank1 message in either ligated or sham-operated animals. The accumulation of Shank1 in the PSD was abolished by intrathecal pre-treatment with anisomycin or Shank1 siRNA but not non-target siRNA. The same pre-treatment prevented both of the early signs of pain behavior. Intrathecal pre-treatment with either MK-801 or U0126 similarly prevented the Shank1 accumulation and alleviated both of the behavioral signs of pain.
The early accumulation of Shank1 in the PSD of dorsal horn neurons may be a necessary step in the injury-associated plasticity that in time leads to the development of persistent pain.
Chronic constriction injury; Glutamate receptors; Neuropathic pain; Nociception; Structural proteins; Synaptic plasticity
In this study, we used electron tomography as well as immuno-gold labeling to analyze the morphology and distribution of proteins within PSDs isolated from rats before birth (embryonic day 19) and at post-natal days 2, 21 and 60. Our data provides direct evidence of distinct morphological and compositional differences in PSDs throughout development. Not all PSD components are present at the early stages of development, with a near lack of the scaffolding molecule PSD-95 at E19 and P2. The presence of NR1 and NR2b suggests that PSD-95 is not directly required for clustering of N-methyl-D-aspartic acid (NMDA) receptors in PSDs early in development. α-Actinin is abundant by E19 suggesting it is a core structural component of the PSD. Both α and β isoforms of Ca2+/calmodulin-dependent protein kinase II (CaMKII) are present early on, but then rise in labeling density approximately four-fold by P21. Of all the molecules studied, only calmodulin (CaM) was found in higher abundance early in PSD development and then fell in amount over time. Spatial analysis of the immuno-gold label shows a non-random distribution for all the proteins studied, lending support to the idea that the PSD is systematically assembled in an organized fashion. Morphological data from electron tomography shows that the PSD undergoes major structural changes through out development.
synapse; electron tomography; synaptogenesis; CaMKII; dendritic spine; immuno-gold labeling
Recent neuroimaging and postmortem studies have demonstrated abnormalities in glutamatergic transmission in major depression. Glutamate NMDA (N-methyl-D-aspartate) receptors are one of the major mediators of excitatory neurotransmission in the central nervous system. At synaptic sites, NMDA receptors are linked with postsynaptic density protein-95 (PSD-95) that plays a key role in mediating trafficking, clustering, and downstream signaling events, following receptor activation. In this study, we examined the expression of NMDA receptor subunits NR1, NR2A, and NR2B as well as PSD-95 in the anterior prefrontal cortex (PFC) using Western blot method. Cortical samples were obtained from age, gender and postmortem interval matched depressed and psychiatrically healthy controls. The results revealed that there was a reduced expression of the NMDA receptor subunits NR2A (−54%) and NR2B (−48%), and PSD-95 protein level (−40%) in the PFC of depressed subjects relative to controls, with no change in the NR1 subunit. The alterations in NMDA receptor subunits, especially the NR2A and NR2B, as well as PSD-95 suggest an abnormality in the NMDA receptor signaling in the PFC in major depression. Our findings in conjunction with recent clinical, cellular, and neuroimaging studies further implicate the involvement of glutamate neurotransmission in the pathophysiology of depression. This study provides additional evidence that NMDA receptor complex is a target for discovery of novel antidepressants.
major depressive disorder; glutamate receptors; postmortem; postsynaptic density protein; anterior prefrontal cortex
Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.
Converging evidence suggests too few activation-ready N-methyl-d-aspartic acid (NMDA) receptor complexes in the postsynaptic density in schizophrenia. Postsynaptic density protein 95 (PSD95), Synaptic GTPase-activating protein (SynGAP), and Multiple PDZ domain protein (MUPP1) are integral components of the NMDA receptor signaling complex, and help facilitate signaling, trafficking, and stabilization. We hypothesized that deficits involving these molecules may contribute to the pathophysiology of schizophrenia. To test our hypothesis, we measured protein expression of PSD95, SynGAP, and MUPP1 in the anterior cingulate cortex and dorsolateral prefrontal cortex. We found decreased PSD95 expression in the anterior cingulate cortex. Antipsychotic medication analyses showed decreased SynGAP expression in the anterior cingulate cortex in patients off medication when analyzed against our comparison group. These data suggest that NMDA receptor complex formation, localization, and downstream signaling may be abnormal in schizophrenia.
MUPP1; N-methyl-d-aspartic acid receptor; PSD95; schizophrenia; SynGAP
Kalirin, a multifunctional Rho GDP/GTP exchange factor, plays a vital role in cytoskeletal organization, affecting process initiation and outgrowth in neurons. Through alternative splicing, the Kalirin gene generates multiple functionally distinct proteins. Kalirin-7 (Kal7) is the most prevalent isoform in the adult rat hippocampus; it terminates with a PDZ binding motif, is localized to the post-synaptic density, interacts with PSD95 and causes the formation of dendritic spines when over-expressed in pyramidal neurons. Levels of Kal7 are low in the dendrites of hippocampal aspiny interneurons. In these interneurons, Kal7 is localized to the postsynaptic side of excitatory synapses onto dendritic shafts, overlapping clusters of PSD95 and NMDA receptor subunit NR1. Selectively decreasing levels of Kal7 decreases the density of PSD95 positive, bassoon positive clusters along the dendritic shaft of hippocampal interneurons. Over-expression of Kal7 increases dendritic branching, inducing formation of spine-like structures along the dendrites and on the soma of normally aspiny hippocampal interneurons. Essentially all of the spine-like structures formed in response to Kal7 are apposed to VGLUT1 positive, bassoon positive presynaptic endings; GAD positive, VGAT positive inhibitory endings are unaffected. Almost every Kal7 positive dendritic cluster contains PSD95 along with NMDA (NR1) and AMPA (GluR1 and GluR2) receptor subunits. Kal7-induced formation of spine-like structures requires its PDZ binding motif, and interruption of interactions between the PDZ binding motif and its interactors decreases Kal7-induced formation of spine-like structures. Kal7 thus joins Shank3 and GluR2 as molecules whose level of expression at excitatory synapses titrates the number of dendritic spines.
Rho GEF; PSD95; GABA; dendritic growth; synaptogenesis
Synaptic plasticity is considered to be the main mechanism for learning and memory. Excitatory synapses in the cerebral cortex and hippocampus undergo plastic changes during development and in response to electric stimulation. It is widely accepted that this process is mediated by insertion and elimination of various glutamate receptors. In a series of recent investigations on left–right asymmetry of hippocampal CA3–CA1 synapses, glutamate receptor subunits have been found to have distinctive expression patterns that depend on the postsynaptic density (PSD) area. Particularly notable are the GluR1 AMPA receptor subunit and NR2B NMDA receptor subunit, where receptor density has either a supralinear (GluR1 AMPA) or inverse (NR2B NMDAR) relationship to the PSD area. We review current understanding of structural and physiological synaptic plasticity and propose a scheme to classify receptor subtypes by their expression pattern with respect to PSD area.
spines; glutamate; AMPAR; NMDAR; mGluR5; PSD
NMDA receptors are found in both synaptic and extrasynaptic locations on neurons. NMDA receptors also can be found on neurons in early stages prior to synaptogenesis, where they may be involved in migration and differentiation. Extrasynaptic NMDA receptors typically are associated with contacts with adjacent processes such as axons and glia. Extrasynaptic NMDA receptor clusters vary in size and may form associations with scaffolding proteins such as PSD-95 and SAP102. The best-characterized extrasynaptic NMDA receptors contain NR1 and NR2B subunits. Extrasynaptic NMDA receptors may be activated by glutamate spillover from synapses or from ectopic release of glutamate. Consequently, extrasynaptic NMDA receptor activation may occur under different circumstances than that for synaptic NMDA receptors, indicating different functional consequences for the neuron. In some cases, activation of extrasynaptic NMDA receptors may have a negative influence on the neuron, leading to cell damage and death, as may occur in some major diseases of the nervous system.
PSD-95, a membrane-associated guanylate kinase (MAGUK), is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNAi knockdown of PSD-95 leads to loss of entire patches of PSD material, and EM tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA, but not NMDA receptor type structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.
PSD-95; molecular conformation; postsynaptic density; hippocampal spines; RNAi; EM tomography; Immuno-EM
PSD-95, a principal scaffolding component of the post-synaptic density, is targeted to synapses by palmitoylation where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction.
One mechanism of information storage in neurons is believed to be determined by the strength of synaptic contacts. The strength of an excitatory synapse is partially due to the concentration of a particular type of ionotropic glutamate receptor (AMPAR) in the post-synaptic density (PSD). AMPAR concentration in the PSD has to be plastic, to allow the storage of new memories; but it also has to be stable to preserve important information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which AMPAR can enter, leave and remain in the synapse are unclear. We used Monte Carlo simulations to determine the influence of PSD structure and activity in maintaining homeostatic concentrations of AMPARs in the synapse. We found that, the high concentration and excluded volume caused by PSD molecules result in molecular crowding. Diffusion of AMPAR in the PSD under such conditions is anomalous. Anomalous diffusion of AMPAR results in retention of these receptors inside the PSD for periods ranging from minutes to several hours in the absence of strong binding of receptors to PSD molecules. Trapping of receptors in the PSD by crowding effects was very sensitive to the concentration of PSD molecules, showing a switch-like behavior for retention of receptors. Non-covalent binding of AMPAR to anchored PSD molecules allowed the synapse to become well-mixed, resulting in normal diffusion of AMPAR. Binding also allowed the exchange of receptors in and out of the PSD. We propose that molecular crowding is an important biophysical mechanism to maintain homeostatic synaptic concentrations of AMPARs in the PSD without the need of energetically expensive biochemical reactions. In this context, binding of AMPAR with PSD molecules could collaborate with crowding to maintain synaptic homeostasis but could also allow synaptic plasticity by increasing the exchange of these receptors with the surrounding extra-synaptic membrane.
One of the most accepted theories of information storage in neurons is that it is partially localized in the strength of synaptic contacts. Evidence suggests that at the cellular level, in combination with other cellular mechanisms, this is implemented by increasing or decreasing the concentration of a particular type of membrane molecules. Two opposing mechanisms have to coexist in synapses to allow them to store information. On one hand, synapses have to be flexible, to allow the storage of new memories. On the other hand, synapses have to be stable to preserve previously learned information. Although much is known about the molecular identity of synapses, the biophysical mechanisms by which molecules can enter, leave and remain in the synapse are unclear. Our modeling work uses fundamental biophysical principles to quantify the effects of molecular collisions and biochemical reactions. Our results show that molecular collisions alone, between the diffusing proteins with anchored molecules in the synapse, can replicate known experimental results. Molecular collision in combination with biochemical binding can be fundamental biophysical principles used by synapses for the formation and preservation of memories.
Activity-dependent modification of excitatory synaptic transmission is fundamental for developmental plasticity of the neural circuits and experience-dependent plasticity. Synaptic glutamatergic receptors including AMPA receptors and NMDA receptors (AMPARs and NMDARs) are embedded in the highly organized protein network in the postsynaptic density. Overwhelming data have shown that PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs), as a major family of scaffold proteins at glutamatergic synapses, regulate basal synaptic AMPAR function and trafficking. It is now clear that PSD-MAGUKs have multifaceted functions in terms of regulating synaptic transmission and plasticity. Here we discuss recent advancements in understanding the roles of PSD-95 and other family members of PSD-MAGUKs in synaptic plasticity, both as an anchoring protein for synaptic AMPARs and also as a signaling scaffold for mediating the interaction of the signaling complex and NMDARs.
It has been postulated that alcoholism is associated with abnormalities in glutamatergic neurotransmission. This study examined the density of glutamate NMDA receptor subunits and its associated proteins in the noradrenergic locus coeruleus (LC) in deceased alcoholic subjects. Our previous research indicated that the NMDA receptor in the human LC is composed of obligatory NR1 and regulatory NR2C subunits. At synapses, NMDA receptors are stabilized through interactions with postsynaptic density protein (PSD-95). PSD-95 provides structural and functional coupling of the NMDA receptor with neuronal nitric oxide synthase (nNOS), an intracellular mediator of NMDA receptor activation. LC tissue was obtained from 10 alcohol-dependent subjects and 8 psychiatrically healthy controls. Concentrations of NR1 and NR2C subunits, as well as PSD-95 and nNOS, were measured using Western blotting. In addition we have examined tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of norepinephrine. The amount of NR1 was lower in the rostral (−30%) and middle (−41%)portions of the LC of alcoholics as compared to control subjects. No differences in the amounts of NR2C, PSD-95, nNOS and TH were detected comparing alcoholic to control subjects. Lower levels of NR1 subunit of the NMDA receptor in the LC implicates altered glutamate-norepinephrine interactions in alcoholism.
glutamate; NMDA receptor; postsynaptic density protein 95; neuronal nitric oxide synthase; tyrosine hydroxylase; locus coeruleus
NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.
Recent studies have identified synaptic proteins that undergo synapse-to-nucleus translocation in response to neuronal activity that modulate protein synthesis. One such translational regulatory protein of the postsynaptic density (PSD) is AIDA-1d, which binds to PSD-95 via its C-terminus. Activation of synaptic NMDA receptors induces the cleavage of AIDA-1d, and the N-terminus is then shuttled to nuclear Cajal bodies where it plays a role in regulating global protein synthesis. Chronic ethanol exposure has been shown to increase the synaptic clustering of NMDA receptors and PSD-95. Here, we tested the hypotheses that AIDA-1d regulates chronic ethanol-induced increases in synaptic NMDA receptor expression. As reported, we found that AIDA-1 was highly enriched in dendritic spines and co-localized with PSD-95. Acute NMDA treatment increased AIDA-1 colocalization with p80 coilin, a marker of Cajal bodies. Chronic treatment (4 day) of cultures with ethanol (25 – 100 mM) or with the NMDA receptor antagonist AP-V (50 µM) enhanced the clustering of AIDA-1 at synaptic sites. However, chronic ethanol treatment (50 mM) in the presence of the NMDA receptor agonist NMDA (2.5 µM) prevented this increase. Surprisingly, PSD-95 did not seem to play a role in the synaptic distribution of AIDA-1 as this distribution was not affected by declustering PSD-95 from synapses in response to inhibition of palmitoylation. We found that lentiviral knockdown of AIDA-1d did not affect protein expression levels of NMDA receptor subunits GluN1, GluN1 C2’, or GluN2B. The results of this study demonstrate that synaptic AIDA-1 expression is enhanced by chronic ethanol exposure that can be prevented by concurrent stimulation of NMDA receptors. In addition, we found that the association of AIDA-1 with PSD-95 is not required for its localization to the PSD. Moreover, we found that AIDA-1 does not regulate protein expression levels or alternative splicing of the GluN1 subunit of NMDA receptors.
AIDA-1, chronic ethanol, synapse-to-nucleus signaling; NMDA receptors; Cajal bodies; PSD-95
Peripheral inflammation or nerve injury induces a primary afferent barrage into the spinal cord, which can cause N-methyl -aspartate (NMDA) receptor-dependent alterations in the responses of dorsal horn sensory neurons to subsequent afferent inputs. This plasticity, such as “wind-up” and central sensitization, contributes to the hyperexcitability of dorsal horn neurons and increased pain-related behavior in animal models, as well as clinical signs of chronic pain in humans, hyperalgesia and allodynia. Binding of NMDA receptor subunits by the scaffolding protein postsynaptic density protein-95 (PSD-95) can facilitate downstream intracellular signaling and modulate receptor stability, contributing to synaptic plasticity. Here, we show that spinal delivery of the mimetic peptide Tat-NR2B9c disrupts the interaction between PSD-95 and NR2B subunits in the dorsal horn and selectively reduces NMDA receptor-dependent events including wind-up of spinal sensory neurons, and both persistent formalin-induced neuronal activity and pain-related behaviors, attributed to central sensitization. Furthermore, a single intrathecal injection of Tat-NR2B9c in rats with established nerve injury-induced pain attenuates behavioral signs of mechanical and cold hypersensitivity, with no effect on locomotor performance. Thus, uncoupling of PSD-95 from spinal NR2B-containing NMDA receptors may prevent the neuronal plasticity involved in chronic pain and may be a successful analgesic therapy, reducing side effects associated with receptor blockade.
Regulation of NMDA receptor (NMDAR) activity by desensitization is important in physiological and pathological states; NMDAR desensitization contributes in shaping synaptic responses and may be protective by limiting calcium influx during sustained glutamate insults. We previously reported that glycine-independent desensitization decreases during hippocampal neuronal development, correlating with NMDAR synaptic localization and association with post-synaptic density 95 (PSD-95). PSD-95/Discs large/zona occludens (PDZ)-1,2 domains of PSD-95 bind to the C-terminus of NMDAR NR2 subunits. The role of PSD-95 in anchoring signaling proteins near NMDARs is well documented. To determine if PSD-95-induced changes in NMDAR desensitization occur because of direct binding to NR2 or due to recruitment of regulatory proteins, we tested the effects of various PSD-95 constructs on NMDAR currents in human embryonic kidney 293 (HEK293) cells and neurons. In HEK cells, wild-type PSD-95 significantly reduced wild-type NMDAR desensitization without altering currents of NMDARs containing NR2A-S1462A, a mutation that abolishes PSD-95 binding. The PSD-95 N-terminus truncated after the PDZ1-2 domains was sufficient for this effect in neurons with low endogenous PSD-95 levels; in NMDAR-expressing HEK cells, the effect persisted when PSD-95 multimerization was eliminated. Moreover, other PSD-95 family members with highly homologous PDZ1-2 domains significantly reduced NMDAR desensitization. In mature neurons, disruption of PSD-95/NMDAR interaction through protein kinase C (PKC) activation increased desensitization to levels found in immature neurons, and this effect was not due to PKC direct regulation of NMDAR activity. We conclude that direct binding of PSD-95 increases stability of NMDAR responses to agonist exposure in neuronal and non-neuronal cells.
Synaptic loss underlies the memory deficit of Alzheimer's disease (AD). The molecular mechanism is elusive; however, excitatory synapses organized by the postsynaptic density (PSD) have been used as targets for AD treatment. To identify pathological entities at the synapse in AD, synaptic proteins were screened by quantitative proteomic profiling. The critical proteins were then selected for immunoblot analysis. The glutamate receptors N-methyl-d-aspartate (NMDA) receptor 1 and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor 2 (GluR2) were substantially lost; specifically, the loss of GluR2 was up to 40% at PSD in AD. Shank proteins, the organizers of these glutamate receptors at excitatory synapses, were dramatically altered in AD. The level of Shank2 was increased, whereas the protein level of Shank3 was decreased. Further, the Shank3 protein was modified by ubiquitin, indicating that abnormal activity of the ubiquitin–proteasome system may lead to Shank3 degradation in AD. Our findings suggest that disruption of glutamate receptors at the Shank-postsynaptic platform could contribute to destruction of the PSD which underlies the synaptic dysfunction and loss in AD.
Alzheimer's; Glutamate receptor; Synapse; Postsynaptic density; Shank; Proteomic
Src family protein kinases (SFKs)-mediated tyrosine-phosphorylation regulates N-methyl-D-aspartate (NMDA) receptor synaptic function. Some members of the membrane-associated guanylate kinase (MAGUK) family of proteins bind to both SFKs and NMDA receptors, but it is unclear whether the MAGUK family of proteins is required for SFKs-mediated tyrosine-phosphorylation of the NMDA receptors. Here, we showed by co-immunoprecipitation that PSD-93, a member of the MAGUK family of proteins, interacts with the NMDA receptor subunits NR2A and NR2B as well as with Fyn, a member of the SFKs, in mouse cerebral cortex. Using a biochemical fractionation approach to isolate subcellular compartments revealed that the expression of Fyn, but not of other members of the SFKs (Lyn, Src, and Yes), was significantly decreased in synaptosomal membrane fractions derived from the cerebral cortex of PSD-93 knockout mice. Interestingly, we found that PSD-93 disruption causes reduction of tyrosine-phosphorylated NR2A and NR2B in the same fraction. Moreover, PSD-93 deletion markedly blocked the SFKs-mediated increase in tyrosine-phosphorylated NR2A and NR2B through the protein kinase C pathway after induction with 4β-PMA in cultured cortical neurons. Our findings indicate that PSD-93 appears to mediate tyrosine-phosphorylation of the NMDA receptors and synaptic localization of Fyn.
Src family tyrosine kinases; Fyn; MAGUK; PSD-95; NR2A; NR2B
The most common causes of neurodegenerative dementia include Alzheimer’s disease (AD), dementia with Lewy bodies (DLB), and frontotemporal dementia (FTD). We believe that, in all 3, aggregates of pathogenic proteins are pathological substrates which are associated with a loss of synaptic function/plasticity. The synaptic plasticity relies on the normal integration of glutamate receptors at the postsynaptic density (PSD). The PSD organizes synaptic proteins to mediate the functional and structural plasticity of the excitatory synapse and to maintain synaptic homeostasis. Here, we will discuss the relevant disruption of the protein network at the PSD in these dementias and the accumulation of the pathological changes at the PSD years before clinical symptoms. We suggest that the functional and structural plasticity changes of the PSD may contribute to the loss of molecular homeostasis within the synapse (and contribute to early symptoms) in these dementias.
Alzheimer’s disease; dementia with Lewy bodies; frontotemporal dementia; postsynaptic density
The N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) are ionotropic glutamate receptors responsible for excitatory neurotransmission in the brain. These excitatory synapses are found on dendritic spines, with the abundance of receptors concentrated at the postsynaptic density (PSD). We utilized two genetic mouse models, the serine racemase knockout (SR−/−) and the glycine transporter subtype 1 heterozygote mutant (GlyT1+/−), to determine how constitutive NMDAR hypo- and hyperfunction, respectively, affect the glutamate receptor composition of the PSD in the hippocampus and prefrontal cortex (PFC). Using cellular fractionation, we found that SR−/− mice had elevated protein levels of NR1 and NR2A NMDAR subunits specifically in the PSD-enriched fraction from the hippocampus, but not from the PFC. There were no changes in the amounts of AMPAR subunits (GluR1, GluR2), or PSD protein of 95kDa (PSD95) in either brain region. GlyT1+/− mice also had elevated protein expression of NR1 and NR2A subunits in the PSD, as well as an increase in total protein. Moreover, GlyT1+/− mice had elevated amounts of GluR1 and GluR2 in the PSD, and higher total amounts of GluR1. Similar to SR−/− mice, there was no protein changes observed in the PFC. These findings illustrate the complexity of synaptic adaptation to altered NMDAR function.
serine racemase; glycine transporter; postsynaptic density; NMDA receptor; AMPA receptor; schizophrenia
The N-methyl-D-aspartate receptors are key mediators of excitatory transmission and are implicated in many forms of synaptic plasticity. These receptors are heterotetrameres consisting of two obligatory NR1 and two regulatory subunits, usually NR2A or NR2B. The NR2B subunits are abundant in the early postnatal brain, while the NR2A/NR2B ratio increases during early postnatal development. This shift is driven by NMDA receptor activity. A functional interplay of the Low Density Lipoprotein Receptor Related Protein 1 (LRP1) NMDA receptor has already been reported. Such abilities as interaction of LRP1 with NMDA receptor subunits or its important role in tPa-mediated NMDA receptor signaling were already demonstrated. Moreover, mice harboring a conditional neuronal knock-out mutation of the entire Lrp1 gene display NMDA-associated behavioral changes. However, the exact role of LRP1 on NMDA receptor function remains still elusive.
To provide a mechanistic explanation for such effects we investigated whether an inactivating knock-in mutation into the NPxY2 motif of LRP1 might influence the cell surface expression of LRP1 and NMDA receptors in primary cortical neurons. Here we demonstrate that a knock-in into the NPxY2 motif of LRP1 results in an increased surface expression of LRP1 and NR2B NMDA receptor subunit due to reduced endocytosis rates of LRP1 and the NR2B subunit in primary neurons derived from LRP1ΔNPxY2 animals. Furthermore, we demonstrate an altered phosphorylation pattern of S1480 and Y1472 in the NR2B subunit at the surface of LRP1ΔNPxY2 neurons, while the respective kinases Fyn and casein kinase II are not differently regulated compared with wild type controls. Performing co-immunoprecipitation experiments we demonstrate that binding of LRP1 to NR2B might be linked by PSD95, is phosphorylation dependent and this regulation mechanism is impaired in LRP1ΔNPxY2 neurons. Finally, we demonstrate hyperactivity and changes in spatial and reversal learning in LRP1ΔNPxY2 mice, confirming the mechanistic interaction in a physiological readout.
In summary, our data demonstrate that LRP1 plays a critical role in the regulation of NR2B expression at the cell surface and may provide a mechanistic explanation for the behavioral abnormalities detected in neuronal LRP1 knock-out animals reported earlier.
LRP1; NPxY2 motif; NMDA receptor; NR1; NR2B receptor subunit; PSD95; Cell surface expression
Homeostatic chemokines, such as CXCL12, can affect neuronal activity by the regulation of inhibitory and excitatory neurotransmission, but the mechanisms involved are still undefined. Our previous studies have shown that CXCL12 protects cortical neurons from excitotoxicity by promoting the function of the gene-repressor protein Rb, which is involved in the recruitment of chromatin modifiers (such as histone deacetylases (HDACs)) to gene promoters. In neurons, Rb controls activity-dependent genes essential to neuronal plasticity and survival, such as the N-methyl--aspartic acid (NMDA) receptor's subunit NR2B, the expression of which in the tetrameric ion channel largely affects calcium signaling by glutamate. In this study, we report that CXCL12 differentially modulates intracellular responses after stimulation of synaptic and extrasynaptic NMDA receptors, by a specific regulation of the NR2B gene that involves HDACs. Our results show that CXCL12 selectively inhibits NR2B expression in vitro and in vivo altering NMDA-induced calcium responses associated with neuronal death, while promoting prosurvival pathways that depend on stimulation of synaptic receptors. Along with previous studies, these findings underline the role of CXCL12/CXCR4 in the regulation of crucial components of glutamatergic transmission. These novel effects of CXCL12 may be involved in the physiological function of the chemokine in both developing and mature brains.
chemokine; neuron; CXCR4; cell death; calcium
The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiqutination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis.
Dendritic pruning and loss of synaptic contacts are early events in many neurodegenerative diseases. These effects are dynamic and appear to differ mechanistically from the cell death process. Cannabinoids modulate synaptic activity and afford protection in some neurotoxicity models. We investigated the effects of cannabinoids on activity-induced changes in the number of synapses between rat hippocampal neurons in culture. Morphology and synapses were visualized by confocal imaging of neurons expressing DsRed2 and postsynaptic density protein 95 fused to enhanced green fluorescent protein (PSD95-GFP). Reducing the extracellular Mg2+ concentration to 0.1 mM for 4 hr induced intense synaptic activity that decreased the number of PSD95-GFP puncta by 45 ± 13 %. Synapse loss was an early event, required activation of NMDA receptors and was mediated by the ubiquitin-proteasome pathway. The cannabinoid receptor full agonist (R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl] pyrrolo-[1,2,3-de]-1,4-benzoxazin-6-yl](1-napthalenyl)methanone monomethanesulfonate (WIN55,212-2; EC50=2.5±0.5 nM) and the partial agonist Δ9-tetrahydrocannabinol (THC; EC50=9±3 nM) inhibited PSD loss in a manner reversed by the CB1 receptor antagonist rimonabant. The protection was mimicked by inhibition of presynaptic Ca2+ channels and WIN55,212-2 did not prevent PSD loss elicited by direct application of glutamate, suggesting a presynaptic mechanism. Prolonged exposure to WIN55,212-2, but not THC, desensitized the protective effect. Treating cells that had undergone PSD loss with WIN55,212-2 reversed the loss and enabled recovery of a full compliment of synapses. The modulation of synaptic number by acute and prolonged exposure to cannabinoids may account for some of the effects of these drugs on the plasticity, survival and function of neural networks.