Glutamate receptor IIA and Glutamate receptor IIB
Genes that specify cell fate can influence multiple aspects of
neuronal differentiation, including axon guidance, target
selection and synapse formation. Mutations in the unc-42
gene disrupt axon guidance along the C. elegans ventral
nerve cord and cause distinct functional defects in sensory-locomotory
neural circuits. unc-42
encodes a novel homeodomain protein that specifies the fate
of three classes of neurons in the Caenorhabditis elegans
nervous system: the ASH polymodal sensory neurons; the
AVA, AVD and AVE interneurons, which mediate repulsive
sensory stimuli to the nematode head and anterior body, and
a subset of motor neurons that innervate head and body-wall
muscles. The UNC-42 sequence contains a paired type homeodomain, most closely related to C. elegans CEH-10 and UNC-4. The homeodomain of UNC-42 is 68% identical to
CEH-10 and 65% identical to UNC-4. UNC-42 exhibits the
same degree of similarity to a number of homeoproteins of the
paired-like classes from other species, including the vertebrate
proteins, Cart-1, Phox2a and Arx -- all Aristalless type homeodomain proteins.
unc-42 is required for the expression of cell-surface
receptors that are essential for the mature function of these
neurons. In mutant animals, the ASH sensory neurons fail
to express SRA-6 and SRB-6, putative chemosensory
receptors. The AVA, AVD and AVE interneurons and RME
and RMD motor neurons of unc-42 mutants similarly fail to
express the GLR-1 glutamate receptor (The predicted GLR-1 protein is roughly 40% identical to mammalian AMPA-class glutamate receptor (GluR) subunits). These results show
that unc-42 performs an essential role in defining neuron
identity and contributes to the establishment of neural
circuits in C. elegans by regulating the transcription of
glutamate and chemosensory receptor genes (Baran, 1999).
The first three unc-42 alleles were identified based on the
uncoordinated (Unc) phenotype conferred by the mutations. Although mutant worms can move forward or
backward spontaneously, this movement is slow and irregular.
Mutant worms tend to kink ventrally and their head movement
is restricted. These defects are likely to be the result of neural
defects because muscle development and morphology are
normal in unc-42 mutants.
In additional assays for behavioral defects, it was found that
unc-42 mutants also exhibit severe defects in response to
mechanical stimuli to the nematode head and anterior body.
unc-42 mutants exhibit a distinctive mechanosensory (Mec)
response to light touch along the body: mutant animals fail
to back up when stroked along anterior body regions, but
moved forward normally when touched along the posterior
body. The response to touch at the tip of the nose
(Not) was also severely reduced in the mutants. Only 8% of
unc-42 animals responded to nose touch, compared to
90% for wild-type worms.
Nose touch and body touch are mediated through separate
neural circuits defined by anatomy, cell
killing experiments and genetic analysis. Wild-type animals
respond to light strokes to the anterior part of the body by
backing up; they respond to touch along the posterior body by moving
forward. The ALMR, ALML and AVM neurons sense light
touch along the anterior body, and the PLMR and PLML
neurons sense touch along the posterior body. Response to anterior body
touch requires the function of the AVA and AVD interneurons,
which innervate type-A motor neurons and drive backward
movement, while response to posterior body touch requires the AVB and
PVC interneurons, which innervate type-B motor neurons and
drive forward movement. The response to anterior body touch is dependent
primarily on the AVD interneurons, probably via gap junctions
with the ALM and AVM sensory neurons. The sensory neurons ASH and FLP at the tip of the nose are the primary detectors of touch and the avoidance responses touch generates.
The ASH neurons also sense high osmolarity
and volatile repellents, and may be analogous to vertebrate
nociceptors that detect several pain modalities. Although the ASH and FLP
sensory neurons are able to synapse with many of the same
interneurons as the ALM and AVM
mechanosensory neurons, the response to nose touch employs a
separate mode of synaptic transmission. ASH-mediated response to
nose touch requires GLR-1, an AMPA-type
glutamate receptor expressed by many
postsynaptic targets of ASH and FLP, including
the AVA and AVD interneurons. Mutations in glr-1 block the
response to nose touch, but do not alter avoidance
to body touch or high osmolarity, even though
these responses are also mediated by the AVA and
AVD interneurons. Mutations in unc-42 alter all of these behaviors: in such cases, response to nose touch and anterior body touch are severely diminished, and osmotic avoidance is also reduced (Baran, 1999).
To determine if developmental defects in the
mechanosensory neurons could account for these
behavioral defects, the morphology and differentiation of mutant ALM,
AVM and ASH sensory neurons were examined. UNC-86, a POU homeodomain
protein required to specify ALM, AVM and FLP cell fate, is expressed normally in the mutants. The axon trajectories of the unc-42 and
wild-type ALM and AVM neurons are also indistinguishable from one another.
The ASH neurons and several other sensory neurons with
ciliated endings take up lipophilic fluorescent dyes such as DiI.
When unc-42 mutants are incubated in media containing DiI,
the ASH neurons take up the dye normally, indicating that their
ciliated endings are exposed. Although ASH axonal and
dendritic morphology appear normal in these animals, other markers for ASH differentiation
are not expressed. The ASH neurons of unc-42 mutants
fail to express an srb-6-gfp transgene, while the ADL and ADF sensory neurons express this transgene normally.
ASH neurons of unc-42 mutants also fail to express an sra-6-gfp transgene. sra-6 and
srb-6 encode seven-transmembrane receptors that may function
as chemoreceptors for the detection of volatile repellents (Baran, 1999).
Failures in ASH function could account for behavioral defects
mediated solely by ASH, but cannot account for the defects in
response to body touch or locomotion, or the severity of the nose-touch
defects of unc-42 mutants. If the ASH neurons of wild-type
animals are killed by laser microsurgery, worms can still
respond to nose touch 37% of the time. By contrast, unc-42 mutants respond to nose-touch
stimuli only 10% of the time. These results predict
that cells in addition to ASH contribute to the nose-touch response
defects of the mutants. To determine if unc-42 mutations also disrupts interneuron
function, a test was performed to see if unc-42 mutants express a
transcriptional glr-1-gfp reporter transgene. glr-1 encodes an
AMPA-type glutamate receptor expressed by all interneurons
of the forward and backward locomotory circuit, as well as by
a subset of head motor neurons. No glr-1-gfp expression could be detected in the AVA, AVD and AVE interneurons in living mutants or in mutant
worms that were fixed and stained with an anti-GFP antiserum
to enhance GFP detection. These
interneurons receive synaptic input from ASH and FLP and
provide output to motor neurons that control backward
movement. The cell bodies of the interneurons are still
present in the mutants. However, it could not be determined if the
morphology of the interneuron axons along the ventral cord was
normal in unc-42 mutants because individual markers for these neurons are not available. Thus, the loss of GLR-1 receptor
expression by the AVA, AVD and AVE interneurons contributes to
the severe nose-touch phenotype of unc-42 mutants. Mutations in unc-42 also disrupt glr-1-gfp expression in
the six RMD and two RME motor neurons, all of which innervate
head muscles. The RME neurons are involved in foraging
behavior, and the RMDs mediate a head withdrawal response to
touch along the side of the nose that is also dependent on glr-1. Because head mobility is defective in unc-42 mutants, mutant
animals were not tested for defects in head withdrawal (Baran, 1999).
UNC-42 expression can be detected in several cells as early as 260 minutes into embryogenesis and by comma stage in neurons at high levels in the head. Comma stage is the time when these neurons extend axons and
establish connections. Expression in head neurons
continues into adulthood. Expression of a transcriptional unc-42-gfp
transgene, gmEx104, is abolished in unc-42 mutants,
suggesting that unc-42 regulates its own expression. UNC-42 was strongly expressed in at least 20 pairs
of neurons in the head, including the AVA, AVD and
AVE interneurons, ASH sensory neurons, and RMD and SMB
motor neurons. Other neurons that express high levels of
UNC-42 include the AIN, AVH, AVJ, AVK, RIV, SAA and
SIB interneurons. Low levels of unc-42 expression are detected in hypodermis and additional neurons in the head. Transient
expression of UNC-42 protein is also detected in the DD
motor neurons at hatching and at low levels
in postembryonic ventral cord motor neurons derived from P11. In the tail, unc-42-gfp is strongly expressed in PVT (Baran, 1999).
UNC-42 is expressed by AVA and AVD interneurons, which
transmit stimuli from the ALM and AVM mechansensory
neurons and the ASH and FLP sensory neurons to motor
neurons. To determine whether defects in the interneurons are
responsible both for the sensory and locomotory defects of
unc-42 mutants, an examination was made of the behavior of animals that were mosaic for unc-42 function. The Unc and Mec phenotypes
are separable in the mosaic animals, reflecting distinct sites
of unc-42 function. Mosaic animals could be isolated that were
Unc but not Mec, as well as animals that were Mec but not Unc. AVA and AVD, the interneurons that mediate
backward motion, are derived from the AB.a blastomere. The
AVB and PVC interneurons that control forward movement,
the ventral cord motor neurons that innervate body wall muscles,
and many of the neurons that regulate head movement are all
descendants of the AB.p blastomere.
11/11 mosaic worms that failed to respond to light touch to the
anterior body, but moved normally, had lost wild-type unc-42
gene activity in AB.a. Six of these losses were in
AB.aala, producing mosaic animals that had mutant AVD
interneurons, wild-type AVM and ALM mechanosensory
neurons and wild-type motor neurons. In contrast, 12/12
mosaic worms that were Unc, but responded to light touch, had
lost wild-type unc-42 function in the AB.p lineage.
A severe uncoordinated phenotype is observed only when all
cells derived from AB.p are mutant for unc-42. Animals with
single losses of wild-type gene function in AB.p descendents
are wild type, and the animals with multiple losses in AB.p
descendents exhibit only weak defects in locomotion.
These results are consistent with the results of laser killing
experiments, which showed that most members of a single
motor neuron class or multiple classes must be killed to
generate a severe uncoordinated phenotype. Because motor neurons are generated at multiple points
in the AB.p cell lineage, only early losses in the
lineage would affect gene function in a significant number of
ventral cord motor neurons. In addition, some of the motor
neurons that innervate head muscles and interneurons that
coordinate head movement also express UNC-42 and are
derived from AB.p.
Genetic mosaic analysis supports the hypothesis that unc-42
acts cell-autonomously in the AVD interneurons for the
response to body touch and is likely to act cell-autonomously
in motor neurons for locomotion (Baran, 1999).
Glutamatergic transmission at a principal neuron-interneuron synapse was investigated by dual whole-cell patch-clamp
recording in rat hippocampal slices combined with morphological analysis. Evoked EPSPs with rapid time course (half
duration = 4 ms; 34 degrees C) were generated at multiple synaptic contacts established on the interneuron dendrites close
to the soma. The underlying postsynaptic conductance change shows a submillisecond rise and decay, due to the precise
timing of glutamate release and the rapid deactivation of the postsynaptic AMPA receptors. Simulations based on a
compartmental model of the interneuron indicate that the rapid postsynaptic conductance change determines the shape and
the somatodendritic integration of EPSPs, thus enabling interneurons to detect synchronous principal neuron activity (Geiger, 1997).
Insulin is expressed in discrete regions throughout the brain. Neurons synthesize and release insulin in response to membrane depolarization, and peripheral insulin
penetrates the blood-brain barrier, entering the brain. Insulin receptors are also highly expressed in CNS neurons and localized to
synapses. Since glucose utilization in neurons is largely insulin independent, CNS insulin may be involved in activities
other than the regulation of glucose homeostasis, and recent evidence is consistent with a wide range of neuronal functions for insulin, including neuromodulation,
growth and maturation, neuronal protection, and learning and memory. However, the detailed mechanisms by which
brain insulin modifies neuronal function remains to be determined. Analogous to its function in peripheral tissues, where insulin produces rapid GLUT4 translocation to
the plasma membrane to increase glucose uptake in these cells, insulin rapidly recruits functional GABAA receptors to postsynaptic domains in mature CNS neurons, resulting in a long-lasting enhancement of GABAA
receptor-mediated synaptic transmission. Insulin can regulate the cell surface expression and hence the function of
various other ion channels and neurotransmitter receptors. Hence, insulin may function as an important CNS
neuromodulator by regulating the intracellular trafficking and plasma membrane expression of ion channels and neurotransmitter receptors (Man, 2000).
Redistribution of postsynaptic AMPA subtype glutamate receptors may regulate synaptic
strength at glutamatergic synapses, but the mediation of the redistribution is poorly understood. AMPA receptors undergo clathrin-dependent
endocytosis, which is accelerated by insulin in a GluR2 subunit-dependent manner. Insulin-stimulated endocytosis rapidly decreases AMPA receptor numbers in
the plasma membrane, resulting in long-term depression (LTD) of AMPA receptor-mediated synaptic transmission in hippocampal CA1 neurons. Moreover,
insulin-induced LTD and low-frequency stimulation-induced homosynaptic CA1 LTD are mutually occlusive and are both blocked by
inhibiting postsynaptic clathrin-mediated endocytosis. Thus, controlling postsynaptic receptor numbers through endocytosis may be an important mechanism
underlying synaptic plasticity in the mammalian CNS (Man, 2000).
To elucidate mechanisms that control and execute activity-dependent synaptic
plasticity, AMPA receptors
(AMPA-Rs) with an electrophysiological tag were expressed in rat hippocampal
neurons. Long-term potentiation (LTP) or increased activity of the
calcium/calmodulin-dependent protein kinase II (CaMKII) induce delivery of
tagged AMPA-Rs into synapses. This effect is not diminished by mutating the
CaMKII phosphorylation site on the GluR1 AMPA-R subunit, but is blocked by
mutating a predicted PDZ domain interaction site. These results show that LTP
and CaMKII activity drive AMPA-Rs to synapses by a mechanism that requires the
association between GluR1 and a PDZ domain protein (Hayashi, 2000).
Both acute and chronic changes in AMPA receptor (AMPAR) localization are critical for synaptic formation, maturation, and plasticity. AMPARs are differentially sorted between recycling and degradative pathways following endocytosis. AMPAR sorting occurs in early endosomes and is regulated by synaptic activity and activation of AMPA and NMDA receptors. AMPAR internalization triggered by NMDAR activation is Ca2+-dependent, requires protein phosphatases, and is followed by rapid membrane reinsertion. Furthermore, NMDAR-mediated AMPAR trafficking is regulated by PKA and accompanied by dephosphorylation and rephosphorylation of GluR1 subunits at a PKA site. In contrast, activation of AMPARs (without NMDAR activation) targets AMPARs to late endosomes and lysosomes, independent of Ca2+, protein phosphatases, or PKA. These results demonstrate that activity regulates AMPAR endocytic sorting, providing a potential mechanistic link between rapid and chronic changes in synaptic strength (Ehlers, 2000).
A key feature of synaptogenesis and synapse maturation is the incorporation and stabilization of postsynaptic AMPARs. Activation of NMDARs switches AMPARs from a degradative pathway to a recycling pathway. This switch in AMPAR sorting could provide a mechanism for maintaining AMPARs at synapses that have an adequate amount of NMDAR activity and for removing AMPARs from synapses with insufficient NMDAR activity. Indeed, postsynaptic NMDARs precede AMPARs at many excitatory synapses during development, and the proportion of these NMDAR-only synapses increases when NMDARs are blocked, perhaps due to selective shunting of AMPARs to lysosomal degradation. Conversely, activation of NMDARs may trigger accumulation and stabilization of AMPARs by promoting AMPAR reinsertion. Thus, by controlling the degree of AMPAR recycling and degradation, NMDAR activation may ensure the maintenance or elimination of AMPARs at appropriate synapses (Ehlers, 2000).
Changes in AMPAR phosphorylation state and synaptic localization are two principal mechanisms proposed to account for long-term changes in synaptic strength. During LTD of hippocampal neuron synapses, GluR1 subunits are dephosphorylated on serine 845, and AMPARs redistribute away from synaptic sites, perhaps due to clathrin-dependent endocytosis. Results presented here provide a possible link between GluR1 dephosphorylation, AMPAR membrane trafficking, and LTD. In particular, these findings suggest a signaling pathway whereby activation of NMDARs triggers a protein phosphatase cascade involving PP2B and PP1 that leads to selective dephosphorylation of GluR1 subunits at serine 845. Dephosphorylation of GluR1, possibly in conjunction with dephosphorylation of components of the endocytic machinery or other AMPAR subunits, then results in AMPAR endocytosis, perhaps by promoting interaction with clathrin adaptors or destabilizing interactions with proteins such as NSF or GRIP. Potentiation of synapses may, in some cases, simply be the reverse of this process. Indeed, results here, showing that membrane insertion of AMPARs occurs simultaneously with phosphorylation at serine 845 and is reduced by PKA inhibitors, are consistent with a finding of selective phosphorylation of serine 845 following potentiation of previously depressed synapses. Coordination of AMPAR phosphorylation and dephosphorylation may thus regulate synaptic strength by regulating AMPAR trafficking (Ehlers, 2000).
A number of proteins interact with the C-terminal regions of GluR2 and GluR3. Two distinct interaction domains on the C-terminal of GluR2 (ct-GluR2) have so far been identified: an NSF binding site between residues 844 and
853 and an extreme ct-PDZ binding motif. The PDZ binding motif has been shown to interact with three PDZ domain-containing proteins:
GRIP and ABP (one splice form is also known as GRIP2), which are closely related, and PICK1. The role of GRIP, ABP, and PICK1 interacting with the C-terminal GluR2 was investigated by infusing a ct-GluR2 peptide ('pep2-SVKI') into CA1
pyramidal neurons in hippocampal slices using whole-cell recordings. Pep2-SVKI, but not a control or PICK1 selective peptide, causes AMPAR-mediated EPSC
amplitude to increase in approximately one-third of control neurons and in most neurons following the prior induction of LTD. Pep2-SVKI also blocks LTD;
however, this occurs in all neurons. A PKC inhibitor prevents these effects of pep2-SVKI on synaptic transmission and LTD. A model is proposed in which the
maintenance of LTD involves the binding of AMPARs to PDZ proteins to prevent their membrane reinsertion (reinsertion normally follows rapidly after activity dependent endocytosis). Evidence is presented that PKC regulates AMPAR
reinsertion during dedepression (Daw, 2000).
AMPA-type glutamate receptors (AMPA-Rs) mediate a majority of excitatory synaptic transmission in the brain. In hippocampus, most AMPA-Rs are hetero-oligomers composed of GluR1/GluR2 or GluR2/GluR3 subunits. These AMPA-R forms display different synaptic delivery mechanisms. GluR1/GluR2 receptors are added to synapses during plasticity; this requires interactions between GluR1 and
group I PDZ domain proteins. In contrast, GluR2/GluR3 receptors replace existing synaptic receptors continuously; this occurs only at synapses that already have AMPA-Rs and requires interactions by GluR2 with NSF and group II PDZ domain proteins. The combination of regulated addition and continuous replacement of
synaptic receptors can stabilize long-term changes in synaptic efficacy and may serve as a general model for how surface receptor number is established and maintained (Shi, 2001).
The molecular interactions with GluR2 that may be necessary for the continuous synaptic delivery of receptors were investigated. Several proteins have been identified that interact with the carboxyl terminus of GluR2, including GRIP1/2(ABP), PICK1, and rDLG6. These proteins are all PDZ (PSD-95, DLG, ZO-1) domain-containing proteins, and the carboxyl terminus of GluR2 corresponds to a group II PDZ domain binding ligand. To examine if such interactions are necessary for the synaptic delivery of GluR2, a mutant GluR2 was generated with a tyrosine (Y) added at the end of the carboxyl terminus (+863Y), a mutation that prevents interaction between GluR2 and PDZ domain-containing proteins. Whole-cell recordings from transfected HEK 293 cells indicate that the recombinant receptor made of this mutant is functional and shows inward rectification similar to GluR2(R586Q)-GFP. However, when expressed in neurons, this mutation blocks synaptic delivery of this receptor, since cells expressing GluR2(R586Q, +863Y)-GFP show no change in rectification of AMPA-R-mediated responses. This indicates that the interactions between GluR2 and group II PDZ domain proteins are necessary for its continuous synaptic delivery. In addition, the amplitude of transmission onto these cells is depressed. The suppression of AMPA-R-mediated transmission may be explained as a dominant negative effect caused by GluR2(R586Q,+863Y)-GFP. This protein may still bind to other proteins (e.g., NSF) required for receptor synaptic delivery and thus compete with endogenous receptors for interactions with the delivery machinery and thereby block the delivery arm of the cycling of endogenous synaptic AMPA-Rs. The removal process may continue, leading to a synaptic depression (Shi, 2001).
Homomeric GluR1 receptors require activity, either LTP or increased CaMKII
activity, to be driven into synapses. This process requires
interactions between GluR1 and PDZ domain proteins. Hetero-oligomeric receptors composed of GluR1 and GluR2, which represent the majority of endogenous GluR1 in hippocampus, also require activity for their delivery. These results are consistent with a model in which GluR1
interacts with proteins that restrict hetero-oligomeric GluR1/GluR2 receptors from synaptic delivery. CaMKII
activity may relieve this restriction. Mere relief of such restriction appears not to be sufficient for synaptic
delivery, however, since GluR1 receptors lacking their carboxyl terminus or GluR1 receptors with a point mutation at the PDZ interaction site are not delivered to synapses. Thus, there appear to be additional protein interactions that effect synaptic delivery (Shi, 2001).
These studies provide direct evidence for two distinct mechanisms by which AMPA-Rs can be delivered to synapses. These two mechanisms can contribute to important aspects of synaptic function. GluR1/GluR2 delivery provides additional receptors following plasticity-inducing stimuli thereby effecting synaptic enhancement. These receptors can be delivered to silent synapses, converting them to functional ones. GluR2/GluR3 receptors can continuously replace synaptic receptors. Thus, this second process can act to preserve plastic changes in the face of protein turnover. How can the number of synaptic receptors be maintained during this continuous replacement? One possibility is that several proteins, in addition to GluR1/GluR2 hetero-oligomers, are delivered in tandem to synapses during plasticity. These proteins could serve as placeholders (i.e., 'slots') that could be filled with nonsynaptic GluR2/GluR3 hetero-oligomers if synaptic GluR1/GluR2 or GluR2/GluR3 hetero-oligomers leave the synapse. GluR1/GluR2 hetero-oligomers may leave 'slots' more slowly (in days) compared to GluR2/GluR3 hetero-oligomers (in minutes), thus explaining why expression of GluR2 carboxyl terminus or infusion of G10/pep2m (a short peptide that mimics the predicted interaction site on GluR2 with NSF) depresses transmission only partially and the GluR2 carboxyl terminus does not block LTP at 1 hr. Some of these delivered proteins could also serve as 'slots' for the eventual addition of NMDA-Rs. Delivery of proteins will likely increase the physical size of synaptic contact and could possibly communicate to the presynaptic side eventually leading to the matching of pre- and postsynaptic size and function (Shi, 2001).
Recent studies show that AMPA receptor trafficking is important in synaptic plasticity. However, the signaling controlling this trafficking is poorly understood. Small GTPases have diverse neuronal functions and their perturbation is responsible for several mental disorders. The roles of small GTPases Ras and
Rap in the postsynaptic signaling underlying synaptic plasticity were examined. Ras relays the NMDA receptor and CaMKII signaling that drives synaptic delivery of AMPA receptors during long-term potentiation. In contrast, Rap mediates NMDA-receptro-dependent removal of synaptic AMPA receptors that occurs during long-term depression. Ras and Rap exert their effects on AMPA receptors that contain different subunit composition. Thus, Ras and Rap, whose activity can be controlled by postsynaptic enzymes, serve as independent regulators for potentiating and depressing central synapses (Zhu, 2002).
The cytoplasmic carboxyl tails of AMPA receptor constituent subunits, which show either long or short forms, control the trafficking characteristics of AMPA receptors. AMPA receptors with long cytoplasmic tails (e.g., GluR1 or GluR4) are restricted from synapses and delivered to synapses during activity-induced synaptic enhancement. AMPA-Rs with only short cytoplasmic tails (e.g., GluR2 or GluR3) cycle continuously from nonsynaptic to synaptic sites in an activity independent manner; their number at synapses can be reduced after activity-induced synaptic depression. The results indicate that spontaneous neural activity continuously adds into the synapses AMPA receptors containing long cytoplasmic tails via Ras activity and continuously removes from synapses AMPA receptors containing only short cytoplasmic tails via Rap activity. Similarly, this study argues that LTP adds to synapses AMPA receptors containing long cytoplasmic tails while LTD removes receptors containing only short cytoplasmic tails. These results indicate the existence of a replacement mechanism at synapses that can exchange AMPA receptors containing long cytoplasmic tails with those containing only short cytoplasmic tails, which explains the observation that LTP and LTD can reverse each other. In fact, this replacement has previously been detected and may itself be under some form of regulation. For example, a more robust replacement appears to occur in dissociated neuronal preparations where LTD stimuli lead to rapid removal of AMPA receptors with long cytoplasmic tails. Thus, the rate of receptor replacement and relative number of receptors with long or short cytoplasmic tails at a synapse may control the amount of LTP or LTD available at that synapse (Zhu, 2002).
Proteins that bind to the cytoplasmic tails of AMPA receptors control receptor trafficking and thus the strength of postsynaptic responses. AP2, a clathrin adaptor complex important for endocytosis, associates with a region of GluR2 that overlaps the NSF binding site. NSF is a hexameric ATPase involved generally in membrane fusion events. Peptides used to interfere with NSF binding also antagonize GluR2-AP2 interaction. Using GluR2 mutants and peptide variants that dissociate NSF and AP2 interaction, it has been found that AP2 is involved specifically in NMDA receptor-induced (but not ligand-dependent) internalization of AMPA receptors, and is essential for hippocampal long-term depression (LTD). NSF function, in contrast, is needed to maintain synaptic AMPA receptor responses, but is not directly required for NMDA receptor-mediated internalization and LTD (Lee, 2002).
The PICK1 protein interacts in neurons with the AMPA-type glutamate receptor subunit 2 (GluR2) and with several other membrane receptors via its single PDZ domain. PICK1 also binds in neurons and in heterologous cells to protein kinase Calpha (PKCalpha) and that the interaction is highly dependent on the activation of the kinase. The formation of PICK1-PKCalpha complexes is strongly induced by TPA, and PICK1-PKCalpha complexes are cotargeted with PICK1-GluR2 complexes to spines, where GluR2 is found to be phosphorylated by PKC on serine 880. PICK1 also reduces the plasma membrane levels of the GluR2 subunit, consistent with a targeting function of PICK1 and a PKC-facilitated release of GluR2 from the synaptic anchoring proteins ABP and GRIP. This work indicates that PICK1 functions as a targeting and transport protein that directs the activated form of PKCalpha to GluR2 in spines, leading to the activity-dependent release of GluR2 from synaptic anchor proteins and the PICK1-dependent transport of GluR2 from the synaptic membrane (Perez, 2001).
Recent studies documenting a role for local protein synthesis in synaptic plasticity have lead to interest in the opposing process, protein degradation, as a potential regulator of synaptic function. The ubiquitin-conjugation system identifies, modifies, and delivers proteins to the proteasome for degradation. Both the proteasome and ubiquitin are present in the soma and dendrites of hippocampal neurons. Since the trafficking of glutamate receptors (GluRs) is thought to underlie some forms of synaptic plasticity, whether blocking proteasome activity affects the agonist-induced internalization of GluRs in cultured hippocampal neurons was examined. Treatment with the glutamate agonist AMPA induces a robust internalization of GluRs. In contrast, brief pretreatment with proteasome inhibitors completely prevents the internalization of GluRs. To distinguish between a role for the proteasome and a possible diminution of the free ubiquitin pool, a chain elongation defective ubiquitin mutant (UbK48R) was expressed that causes premature termination of polyubiquitin chains but, importantly, can serve as a substrate for mono-ubiquitin-dependent processes. Expression of K48R in neurons severely diminishes AMPA-induced internalization establishing a role for the proteasome. These data demonstrate the acute (e.g., minutes) regulation of synaptic function by the ubiquitin-proteasome pathway in mammalian neurons (Patrick, 2003).
Long-term maintenance and modification of synaptic strength involve the turnover of neurotransmitter receptors. Glutamate receptors are constitutively and acutely internalized, presumptively through clathrin-mediated receptor endocytosis. cpg2 is a brain-specific splice variant of the syne-1 gene that encodes a protein specifically localized to a postsynaptic endocytotic zone of excitatory synapses. RNAi-mediated CPG2 knockdown increases the number of postsynaptic clathrin-coated vesicles, some of which traffic NMDA receptors, disrupts the constitutive internalization of glutamate receptors, and inhibits the activity-induced internalization of synaptic AMPA receptors. Manipulating CPG2 levels also affects dendritic spine size, further supporting a function in regulating membrane transport. These results suggest that CPG2 is a key component of a specialized postsynaptic endocytic mechanism devoted to the internalization of synaptic proteins, including glutamate receptors. The activity dependence and distribution of cpg2 expression further suggest that it contributes to the capacity for postsynaptic plasticity inherent to excitatory synapses (Cottrell, 2004).
Screens for plasticity-related genes have identified multiple transcripts that encode synaptic proteins, suggesting that genes induced by activity often function in normal synaptic processes. candidate plasticity gene 2 (cpg2) was isolated in a screen for transcripts upregulated by kainic acid-induced seizures in the rat dentate gyrus, and its expression is regulated during development and by sensory experience. cpg2 is a splice variant of the syne-1 gene, a large gene that encodes a protein with an actin binding domain at the N terminus and a nuclear transmembrane domain at the C terminus, separated by a long helical region. The cpg2 transcript is derived from a portion of the separator region, encodes a protein with homologies to dystrophin, and contains motifs predicting a structural function, including several spectrin repeats and coiled coils. Proteins with these motifs often play a central role in organizing protein complexes (Cottrell, 2004).
cpg2 is expressed only in the brain and encodes a protein that localizes specifically to the postsynaptic endocytic zone of excitatory synapses. Evidence is presented that CPG2 is a critical component of the postsynaptic endocytic pathway that mediates both constitutive and activity-regulated glutamate receptor internalization. It is hypothesized that CPG2 is a key component of a specialization that is devoted to the internalization of postsynaptic proteins at synapses capable of plasticity (Cottrell, 2004).
The synapse contains densely localized and interacting proteins that enable it to adapt to changing inputs. A Ca2+-sensitive protein complex is described involved in the regulation of AMPA receptor synaptic plasticity. The complex is comprised of (1) MUPPI, a multi-PDZ domain-containing protein, (2) SynGAP, a synaptic GTPase-activating protein, and (3) the Ca2+/calmodulin-dependent kinase CaMKII. In synapses of hippocampal neurons, SynGAP and CaMKII are brought together by direct physical interaction with the PDZ domains of MUPP1, and in this complex, SynGAP is phosphorylated. Ca2+CaM binding to CaMKII dissociates it from the MUPP1 complex, and Ca2+, entering the cell via the NMDAR, drives the dephosphorylation of SynGAP. Specific peptide-induced SynGAP dissociation from the MUPP1-CaMKII complex results in SynGAP dephosphorylation accompanied by P38 MAPK inactivation, potentiation of synaptic AMPA responses, and an increase in the number of AMPAR-containing clusters in hippocampal neuron synapses. siRNA-mediated SynGAP knockdown confirms these results. These data implicate SynGAP in NMDAR- and CaMKII-dependent regulation of AMPAR trafficking (Krapivinsky, 2004).
Removal of synaptic AMPA receptors is important for synaptic depression. This study characterizes the roles of individual subunits in the inducible redistribution of AMPA receptors from the cell surface to intracellular compartments in cultured hippocampal neurons. The intracellular accumulation of GluR2 and GluR3 but not GluR1 is enhanced by AMPA, NMDA, or synaptic activity. After AMPA-induced internalization, homomeric GluR2 enters the recycling pathway, but following NMDA, GluR2 is diverted to late endosomes/lysosomes. In contrast, GluR1 remains in the recycling pathway, and GluR3 is targeted to lysosomes regardless of NMDA receptor activation. Interaction with NSF plays a role in regulated lysosomal targeting of GluR2. GluR1/GluR2 heteromeric receptors behave like GluR2 homomers, and endogenous AMPA receptors show differential activity-dependent sorting similar to homomeric GluR2. Thus, GluR2 is a key subunit that controls recycling and degradation of AMPA receptors after internalization (Lee, 2004).
Fast excitatory synaptic transmission in the mammalian brain is mediated primarily by AMPA-type glutamate receptors. Recently, the dynamic redistribution of AMPA receptors in and out of synapses has emerged as an important mechanism for certain forms of long-lasting synaptic modification. In the hippocampal CA3-CA1 synapse, net delivery of AMPA receptors to the postsynaptic membrane leads to long-term potentiation (LTP), whereas net removal of AMPA receptors by internalization from the surface seems to underlie long-term depression (LTD) (Lee, 2004 and references therein).
AMPA receptors are heterotetrameric complexes composed of various combinations of four subunits (GluR1-4). In the adult hippocampus, two major subtypes of AMPA receptors exist that contain either GluR1 and GluR2, or GluR2 and GluR3 subunits. GluR4 is mainly expressed early in development. Individual AMPA receptor subunits interact via their cytoplasmic tails with different sets of proteins. These specific protein interactions are believed to regulate the trafficking and synaptic targeting of AMPA receptors (Lee, 2004 and references therein).
Subunit-specific functions governing the synaptic delivery of AMPA receptors have been uncovered by elegant electrophysiological assays in hippocampal slice cultures and corroborated by cell biological studies in dissociated cultures. GluR1 is the key subunit that 'drives' AMPA receptors to the surface and to synapses in response to NMDA receptor stimulation and activation of CaMKII, resulting in synaptic potentiation. GluR2 on the other hand is delivered constitutively to synapses, replacing existing receptors with no change in synaptic strength. In heteromeric receptors, GluR1 acts 'dominantly' over GluR2, whereas GluR2 acts dominantly over GluR3. Thus, in the hippocampus, it is believed that GluR1/2 heteromers are delivered to synapses during activity-dependent synaptic potentiation, such as LTP, whereas GluR2/3 heteromers cycle continuously between the postsynaptic membrane and intracellular compartments (Lee, 2004).
In contrast to synaptic delivery, little is known about the roles of individual subunits in the removal of AMPA receptors from synapses. Earlier studies on endogenous AMPA receptors suggest that both GluR1- and GluR2-containing receptors can undergo inducible internalization upon stimulation with AMPA, NMDA, or insulin, but GluR1 and GluR2 are often coassembled in the same heteromeric receptor, so these studies could not distinguish their subunit-specific roles in endocytosis (Lee, 2004).
After endocytosis, AMPA receptors undergo endosomal sorting like any other internalized membrane protein -- ultimately, they can be recycled back to the surface membrane or degraded in lysosomes. However, it is not known whether the endosomal sorting of AMPA receptor depends on subunit composition or how activity might affect the intracellular fate of specific subunits (Lee, 2004).
One caveat of AMPA receptor internalization studies is that, due to technical reasons, the quantitation of 'internalization' (e.g., by surface biotinylation or antibody feeding assays) does not strictly measure endocytosis per se but rather the amount of surface receptors that have internalized and that remain in intracellular compartments. Because AMPA receptors cycle rapidly between intracellular and plasma membranes, the amount of internalized receptor is strongly affected by the rate of recycling to the surface as well as by the rate of endocytosis. Therefore, the terms 'redistribution to intracellular compartments' or 'intracellular accumulation' are preferred rather than 'internalization' to signify the amount of surface receptor that is redistributed to internal pools. Insofar as it reflects a shift from surface to intracellular compartments, the measure of intracellular accumulation of internalized receptors is still relevant to removal of AMPA receptors from the synapse (Lee, 2004).
In this report, the subunit rules have been investigated that govern the activity-dependent redistribution of surface AMPA receptors to intracellular compartments and that determine the intracellular sorting of receptors after they are internalized. In contrast to inducible synaptic delivery, where GluR1 plays the key role, the GluR2 subunit is the primary determinant of inducible intracellular accumulation of AMPA receptors. GluR2 controls the postendocytic trafficking of internalized AMPA receptors to either recycling or lysosomal degradation pathways, at least in part dependent on its interaction with NSF (Lee, 2004).
Synaptic trafficking of AMPA-Rs, controlled by small GTPase Ras signaling, plays
a key role in synaptic plasticity. However, how Ras signals synaptic AMPA-R
trafficking is unknown. This study shows that low levels of Ras activity stimulate
extracellular signal-regulated kinase kinase (MEK)-p42/44 MAPK (extracellular
signal-regulated kinase [ERK]) signaling, whereas high levels of Ras activity
stimulate additional Pi3 kinase (Pi3K)-protein kinase B (PKB) signaling, each
accounting for ~50% of the potentiation during long-term potentiation (LTP).
Spontaneous neural activity stimulates the Ras-MEK-ERK pathway that drives
GluR2L into synapses. In the presence of neuromodulator agonists, neural
activity also stimulates the Ras-Pi3K-PKB pathway that drives GluR1 into
synapses. Neuromodulator release increases with increases of vigilance.
Correspondingly, Ras-MEK-ERK activity in sleeping animals is sufficient to
deliver GluR2L into synapses, while additional increased Ras-Pi3K-PKB activity
in awake animals delivers GluR1 into synapses. Thus, state-dependent Ras
signaling, which specifies downstream MEK-ERK and Pi3K-PKB pathways,
differentially control GluR2L- and GluR1-dependent synaptic plasticity (Y. Qin, 2005).
The results suggest that Ras signals synaptic insertion of AMPA-Rs via
stimulating phosphorylation of S845 and S831 of GluR1 and S842 of GluR2L.
Because Ras downstream signaling molecules ERK and PKB are unlikely to
directly phosphorylate GluR1 and GluR2L,
other molecules probably exist at synapses to relay the
signaling. Two likely candidates are cAMP-dependent protein kinase (PKA) and
calcium/calmodulin-dependent protein kinase II (CaMKII), since they can
phosphorylate S845 and S831 of GluR1, respectively.
Protein kinase C (PKC) is another putative candidate because it can
phosphorylate S831, as well as S845, albeit to a lesser extent.
However, whether ERK and PKB stimulate PKA,
CaMKII, and/or PKC remains to be examined. In contrast, serine/threonine
kinases Rsk (see RSK) and mTOR-S6K, which relay downstream Ras signaling in nonneuronal
cells, may also serve as the relays. In particular, both Rsk and mTOR are expressed at synapses, and disruption of Rsk and mTOR signaling leads to mental retardation.
Thus, determining the precise functional relationships (i.e., sequential or
parallel, and downstream or upstream) of the signaling molecules involved in Ras
pathways during LTP is central to answer many important questions related to the
mechanisms of synaptic plasticity (Y. Qin, 2005).
Though NMDA-R-dependent forms of synaptic plasticity have been extensively
examined in vitro, little is known about their properties in the intact brain.
Previous studies have shown that both the occurrence and magnitude of LTP
induced by electric tetanization stimuli are higher in awake than sleeping
animals. However, the mechanisms
of this state-dependent LTP are unclear, because the LTP-inducing stimuli do not
mimic physiological activity in these states. Both GluR2L
and GluR1 mediate LTP in juvenile and adult animals. This study reports
that synaptic activity in sleeping animals
is sufficient for driving GluR2L but not GluR1 into synapses, whereas synaptic
activity in awake animals drives more GluR2L as well as GluR1 into synapses,
suggesting more synaptic plasticity in awake animals. Based on these
findings, it is proposed that state-dependent physiological factors, such as
neuromodulators, may control the state-dependent plasticity. Indeed,
neuromodulator agonists (for example, histamine, a monoamine neuromodulator)
can drive more GluR2L as well as GluR1 into synapses, by
stimulating Ras signaling. These results are consistent with the previous
findings that neuromodulators, whose release increases in general during the
awake behavioral state, stimulate ERK and Pi3K signaling and potentiate LTP. It remains to be determined whether other state-dependent factors (i.e., neuronal firing patterns, hormones, and neurotrophic factors) regulate synaptic plasticity and how these factors interact in the intact brain (Y. Qin, 2005).
Memory consolidation seems to occur during sleep and waking, while learning
occurs in the conscious state.
It is believed that the learning and memory processes require synaptic
plasticity. This study shows that synaptic potentiation is present in both sleeping
and awake states. Interestingly, synaptic plasticity in sleeping and awake
states is controlled by different levels of Ras signaling and mediated by
trafficking of distinct AMPA-Rs. The obvious puzzles are whether and how
Ras-regulated, subunit-specific AMPA-R trafficking correlates with the different
forms of memory consolidation and learning (e.g., declarative vs. procedural or
explicit vs. implicit). Manipulating Ras signaling and trafficking of AMPA-Rs in
intact animals (e.g., in vivo expression of Ras mutants and GluRct-GFP)
during different behavioral states (e.g., slow-wave sleep, REM sleep, quiescent alert, and active exploring) and monitoring changes in learning and memory behavior promise to reveal new insights into these pivotal questions (Y. Qin, 2005).
NMDA receptors (NMDARs) control bidirectional synaptic plasticity by regulating postsynaptic AMPA receptors (AMPARs). NMDAR activation can have differential effects on AMPAR trafficking, depending on the subunit composition of NMDARs. In mature cultured neurons, NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1, primarily by regulating its surface insertion. In mature neurons, NR2B is coupled to inhibition rather than activation of the Ras-ERK pathway, which drives surface delivery of GluR1. Moreover, the synaptic Ras GTPase activating protein (GAP) SynGAP is selectively associated with NR2B-NMDARs in brain and is required for inhibition of NMDAR-dependent ERK activation. Preferential coupling of NR2B to SynGAP could explain the subtype-specific function of NR2B-NMDARs in inhibition of Ras-ERK, removal of synaptic AMPARs, and weakening of synaptic transmission (Kim, 2005 ).
The ERK1/2 signaling pathway is activated by calcium influx through NMDARs and plays an important role in synaptic plasticity and cell survival. NMDAR-dependent ERK activation involves the small GTPase Ras, which is stimulated by specific guanine nucleotide exchange factors (GEFs) and inhibited by GTPase activating proteins (GAPs). The RasGEF RasGRF1 is reported to bind directly to the NR2B subunit of NMDARs. SynGAP, a RasGAP highly enriched in the postsynaptic density (PSD), can associate with NMDARs through binding to PSD-95 family proteins. The exact function of these Ras regulatory proteins in synaptic plasticity has not been established, and how they are functionally coupled to NMDARs remains unclear (Kim, 2005).
Altered AMPAR trafficking has emerged as a major postsynaptic mechanism for the expression of synaptic plasticity. A prevailing model is that NMDAR-dependent LTP is mediated by the surface insertion and synaptic delivery of GluR1, that is driven by CaM kinase II and the Ras-ERK pathway. In contrast, LTD is supposed to result, at least in part, from the removal of synaptic AMPARs by the increased endocytosis and/or reduced recycling of GluR2/3 subunits (Kim, 2005).
This study investigates the links between NMDAR subtypes, Ras-ERK signaling, and AMPAR trafficking. NR2A and NR2B are found to have antagonistic actions on Ras-ERK activation and AMPAR distribution in mature neurons. NR2A-NMDARs promote, whereas NR2B-NMDARs inhibit, the surface expression of GluR1 -- primarily by regulating GluR1 surface insertion. Potentially accounting for this difference is that NR2B is coupled to the inhibition rather than the activation of the Ras-ERK pathway. This functional coupling is correlated with the specific biochemical association of SynGAP with NR2B-NMDARs (Kim, 2005).
PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. This study reports protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCa or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. It is suggested that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking (Lu, 2005).
NMDA receptor-dependent long-term potentiation and long-term depression (LTD) involve changes in AMPA receptor activity and postsynaptic localization that are in part controlled by glutamate receptor 1 (GluR1) subunit phosphorylation. The scaffolding molecule A-kinase anchoring protein (AKAP)79/150 targets both the cAMP-dependent protein kinase (PKA) and protein phosphatase 2B/calcineurin (PP2B/CaN) to AMPA receptors to regulate GluR1 phosphorylation. Brief NMDA receptor activation leads to persistent redistribution of AKAP79/150 and PKA-RII, but not PP2B/CaN, from postsynaptic membranes to the cytoplasm in hippocampal slices. Similar to LTD, AKAP79/150 redistribution requires PP2B/CaN activation and is accompanied by GluR1 dephosphorylation and internalization. Using fluorescence resonance energy transfer microscopy in hippocampal neurons, it has been demonstrated that PKA anchoring to AKAP79/150 is required for NMDA receptor regulation of PKA-RII localization and that movement of AKAP-PKA complexes underlies PKA redistribution. These findings suggest that LTD involves removal of AKAP79/150 and PKA from synapses in addition to activation of PP2B/CaN. Movement of AKAP79/150-PKA complexes from the synapse could further favor the actions of phosphatases in maintaining dephosphorylation of postsynaptic substrates, such as GluR1, that are important for LTD induction and expression. In addition, these observations demonstrate that AKAPs serve not solely as stationary anchors in cells but also as dynamic signaling components (Smith, 2006).
AMPA-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. An atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with mu2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2mu and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre- and post-synaptic membrane proteins (Kastning, 2007).
Synaptic activity regulates the postsynaptic accumulation of AMPA receptors over timescales ranging from minutes to days. Indeed, the regulated trafficking and mobility of GluR1 AMPA receptors underlies many forms of synaptic potentiation at glutamatergic synapses throughout the brain. However, the basis for synapse-specific accumulation of GluR1 is unknown. This study reports that synaptic activity locally immobilizes GluR1 AMPA receptors at individual synapses. Using single-molecule tracking together with the silencing of individual presynaptic boutons, it was demonstrated that local synaptic activity reduces diffusional exchange of GluR1 between synaptic and extraynaptic domains, resulting in postsynaptic accumulation of GluR1. At neighboring inactive synapses, GluR1 is highly mobile with individual receptors frequently escaping the synapse. Within the synapse, spontaneous activity confines the diffusional movement of GluR1 to restricted subregions of the postsynaptic membrane. Thus, local activity restricts GluR1 mobility on a submicron scale, defining an input-specific mechanism for regulating AMPA receptor composition and abundance (Ehlers, 2007).
Bon, C. L. and Garthwaite, J. (2003). On the role of nitric oxide in hippocampal long-term potentiation. J. Neurosci. 23: 1941-1948. PubMed citation: 12629199
Feil, R., Hofmann, G. and Kleppisch, T. (2005). Function of cGMP-dependent protein kinases in the nervous system. Rev. Neurosci. 16: 23-41. PubMed citation: 15810652
Huang, Y., et al. (2005). S-nitrosylation of N-ethylmaleimide sensitive factor mediates surface expression of AMPA receptors. Neuron 46: 533-540. PubMed citation: 15944123
Puzzo, E., et al. (2005). Amyloid-beta peptide inhibits activation of the nitric oxide/cGMP/cAMP-responsive element-binding protein pathway during hippocampal synaptic plasticity. J. Neurosci. 25: 6887-6897. PubMed citation: 16033898
Serulle, Y., et al. (2007). A GluR1-cGKII interaction regulates AMPA receptor trafficking. Neuron 56(4): 670-88. PubMed citation: 18031684
Wang, H. G., et al. (2005). Presynaptic and postsynaptic roles of NO, cGK, and RhoA in long-lasting potentiation and aggregation of synaptic proteins. Neuron 45: 389-403. PubMed citation: 15694326
Trafficking of AMPA receptors (AMPARs) is regulated by specific interactions of the subunit intracellular C-terminal domains (CTDs) with other proteins, but the mechanisms involved in this process are still unclear. This study found that the GluR1 CTD binds to cGMP-dependent protein kinase II (cGKII) adjacent to the kinase catalytic site. Binding of GluR1 is increased when cGKII is activated by cGMP. cGKII and GluR1 form a complex in the brain, and cGKII in this complex phosphorylates GluR1 at S845, a site also phosphorylated by PKA. Activation of cGKII by cGMP increases the surface expression of AMPARs at extrasynaptic sites. Inhibition of cGKII activity blocks the surface increase of GluR1 during chemLTP and reduces LTP in the hippocampal slice. This work identifies a pathway, downstream from the NMDA receptor (NMDAR) and nitric oxide (NO), which stimulates GluR1 accumulation in the plasma membrane and plays an important role in synaptic plasticity (Serulle, 2007).
NMDAR stimulation activates nNOS and production of NO, which results in cGMP production and cGKII activation. A major mechanism for expression of NMDAR-dependent LTP involves the synaptic insertion of GluR1. This study reports that, following activation by the NMDAR, cGKII binds to GluR1 and phosphorylates S845, leading to an increase of GluR1 in the plasma membrane. Notably, a cGKII dominant-negative inhibitor peptide blocked the cGMP-dependent increase of GluR1 surface expression, prevented the increase in amplitude and frequency of mEPSCs after chemLTP, and strongly reduced LTP in hippocampal slices. These results demonstrate a mechanism in which the NMDAR regulates AMPAR trafficking during LTP via NO and cGKII (Serulle, 2007).
Because NO is produced at postsynaptic sites and can diffuse through lipid membranes, initial studies of NO-dependent plasticity focused on presynaptic NO function through retrograde mechanisms. Some results were controversial, possibly because different methodologies were employed. Indeed, cGMP derivatives only facilitate LTP maximally if briefly applied when the NMDA receptor is active, and deviating protocols would lead to conflicting results. More recently, the use of new NO donors and NOS antagonists (Bon, 2003; Puzzo, 2005), both in vitro and in vivo (Feil, 2005), has demonstrated a role of the NO cascade in synaptic plasticity. Interestingly, as reported here, both the sGC inhibitor ODQ and the cGK inhibitor KT5823 were found to block LTP. Nonetheless, specific molecular mechanisms underlying the effects of NO, in particular in NO control of AMPAR trafficking in LTP, have been wanting. S-nitrosylation of NSF enhances NSF binding to GluR2 and regulates GluR2 surface expression (Huang, 2005). Also, activation of the NO-cGMP-cGKI pathway increases both GluR1 and synaptophysin puncta and the phosphorylation of VASP in hippocampal neurons (Wang, 2005). However, as yet, a specific pathway for NO control of activity-dependent GluR1 trafficking to synapses, an essential component of LTP, has not been reported. The interaction of cGKII with GluR1 reported here, and its consequent effect on GluR1 surface levels, directly link the actions of NO to LTP via GluR1 trafficking (Serulle, 2007).
A physical association of cGKII with GluR1 enables the kinase to phosphorylate GluR1 at S845. This phosphorylation is required for cGMP-dependent GluR1 surface accumulation, since block of the phosphorylation by the S845A GluR1 mutation blocked the surface increase. Phosphorylation of S845 accompanies increases in GluR1 surface levels and is necessary for GluR1 synaptic insertion during LTP. S845 is dephosphorylated during hippocampal LTD, and S845 phosphorylation on its own is sufficient for increase of GluR1 in the extrasynaptic plasma membrane. Thus far only PKA phosphorylation of S845 has been considered, perhaps because it was the initial kinase shown to phosphorylate this site. The present study demonstrates that cGKII activity also phosphorylates S845 (Serulle, 2007).
Increases of surface GluR1 following both PKA and cGKII phosphorylation are restricted to extrasynaptic sites , and AMPAR synaptic incorporation requires at least one additional step, possibly mediated by S818 phosphorylation. Interestingly, although 8-Br-cGMP on its own did not enhance hippocampal synaptic responses, when paired with a weak tetanus that by itself does not enhance responses, 8-Br-cGMP produced an immediate potentiation. This suggests that cGMP can prime the system for potentiation by a weak tetanic stimulation, possibly by increasing the extrasynaptic surface AMPAR population (Serulle, 2007).
The NMDAR and nNOS mutually interact with PSD-95, and Ca2+ fluxes through the NMDAR activate nNOS in this complex to produce NO, which induces sGC to produce cGMP, which activates cGKII. Ca2+ fluxes also stimulate Ca2+-regulated adenylate cyclases, which produce cAMP, which activates PKA, which also phosphorylates S845. PKA binds the A kinase anchoring protein, AKAP79, which in turn binds the PDZ domain scaffolding protein, SAP97, which binds the GluR1 CTD, thus targeting PKA to the GluR1 CTD and facilitating phosphorylation of S845 (Serulle, 2007).
Unlike the SAP97-AKAP-PKA pathway, the NO-cGMP-cGKII pathway does not rely on a scaffold since the kinase binds the receptor directly. Interestingly, a knockin mouse expressing GluR1 that lacks the last 7 aa of its CTD and does not bind SAP97 exhibited normal hippocampal LTP and GluR1 trafficking. This is explained if the NO-cGMP-cGKII pathway phosphorylates S845 in this mutant (Serulle, 2007).
GluR1 interacts with cGKII via auxiliary and core contact CTD sequences that flank S845. Interestingly, a CTD contact sequence resembles an AI domain sequence of cGKII, suggesting that to bind the catalytic domain, GluR1 mimics the AI domain. Also, this receptor-kinase interaction resembles the well-studied CaMKII binding to the NR2B (Serulle, 2007).
In the absence of cGMP, cGKII is inactive. Following NMDAR stimulation, binding of cGMP to cGKII induces a cGKII conformational change that causes AI domain autophosphorylation, AI domain release from the catalytic domain, and elongation of the kinase. The GluR1 CTD binds the newly exposed cGKII catalytic domain, facilitating GluR1 phosphorylation and the increase of surface GluR1. In one model for this increase, S845 phosphorylation promotes GluR1 trafficking to the plasma membrane, perhaps by releasing of GluR1 from a cytosolic retention factor. Alternatively, GluR1 may cycle into and out of the plasma membrane constitutively, and S845 phosphorylation may stabilize the receptor at the neuron surface. With either model, S845 phosphorylation would regulate the size of an extrasynaptic pool from which receptors may be inserted into the synapse during LTP. Such transport may depend on additional GluR1 phosphorylation. Because a highly selective peptide block of cGKII strongly reduces LTP, such an increase in an extrasynaptic receptor pool is likely to be a requirement for the synaptic potentiation associated with LTP. The present work demonstrates that the NMDAR can control the size of such a receptor pool, acting through nNOS, NO, and cGMP production and the activation of cGKII (Serulle, 2007).
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Glutamate receptor IIA and Glutamate receptor IIB:
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