H . Tanaka et al. Brain Research 886 2000 190 –207
191
21
a rise in intracellular free Ca , which activates a number
[42,52] voltage-dependent block by polyamines [13,32] of proteases, phospholipases and endoncleases, by gene-
and single channel conductance [141] of recombinant ration of free radicals that destroy cellular membranes by
AMPARs expressed in Xenopus oocytes and mammalian lipid peroxidations for reviews, see [27,29,122,135] and
cells. by induction of apoptosis [29]. Possible mechanisms by
AMPARs are ligand-gated channels and, by analogy to
21
which glutamate could elicit a rise in intracellular Ca the nicotinic acetylcholine receptor, are thought to be
21
include: 1 activation of Ca -permeable AMPA a-
tetrameric or pentameric assemblies arranged around a amino-3-hydroxy-5-methyl-4-isoazole-proprionic
acid- central aqueous pore.
type glutamate receptors AMPARs [146,148]; 2 activa- The current model of GluR subunit topology in the
tion of
group 1
metabotropic glutamate
receptors membrane includes: 1 a large extracellular amino-termi-
mGluRs, which are positively linked to inositol phos- nal domain; 2 three transmembrane-spanning domains
21
phates; 3 activation of voltage-sensitive Ca channels;
TM1, TM3 and TM4; 3 a fourth amphipathic segment and or 4 de-activation of extrusion and or sequestration
TM2 that forms a channel-lining reentrant hairpin loop,
1
systems [99]. similar in structure to the pore-forming region of K
21
Until recently, AMPARs were thought to be Ca -
channels [158]; 4 a binding domain for agonists formed impermeable. It is now well established that the presence
from segments of the amino-terminal domain and extracel- of the GluR2 subunit in heteromeric AMPAR assemblies
lular loop [138]; and 5 an intracellular C-terminal
21 21
governs the permeability of AMPARs to Ca and Zn
. domain. The dominance of the GluR2 subunit in determin-
21
In the adult mammalian central nervous system under ing permeability to Ca
and other divalent ions is physiological conditions, the vast majority of cells and
attributed to the presence of a positively charged arginine
21
tissues express GluR2-containing, Ca -impermeable AM-
R in place of a glutamine Q residue within TM2, which PARs. Thus, a change in the level of GluR2 expression
forms the selectivity filter of AMPARs [21,57]. Although would be expected to have significant physiological conse-
most hippocampal
neurons express
predominantly quences. The relative expression of GluR2 subunit mRNA
heteromeric AMPARs, they may also express GluR1 and protein in neurons is not static but is regulated in a
homomers [80,157]. cell-specific manner during development [108] and may be
AMPARs are differentially expressed throughout neu- remodeled after seizures [38,113,114] or ischemic insult
rons of the mammalian central nervous system. Studies [38,44,111,114] and by administration of anti-psychotics
involving patch-clamp recording combined with RT-PCR
21
[35] drugs of abuse [36,102] or corticosteroids [93]. Ca -
reverse transcriptase-polymerase chain reaction demon-
21
permeable AMPARs are implicated in synaptogenesis and strate that AMPAR permeability to Ca
varies inversely formation of neuronal circuitry, particularly at times and in
with abundance of GluR2 mRNA in a wide range of cell
21
cells in which NMDAR expression is low. Ca influx via
types Table 1. Excitatory principal neurons such as
21
Ca -permeable AMPARs is thought to play a critical role
hippocampal [12] and neocortical [60] pyramidal cells and in growth cone movement and experience-dependent prun-
dentate gyrus granule cells [42] express abundant GluR2
21
ing of synaptic connections during early development [81]. mRNA and exhibit low AMPAR Ca
-permeability. Thus, This article reviews recent studies that address transcrip-
a change in GluR2 expression would be expected to have tional and translation regulation and targeting and traffick-
significant physiological consequences. ing of the AMPAR subunit GluR2 under physiological and
pathological conditions, with a particular emphasis on 2.1. GluR2 RNA editing
transcriptional regulation of GluR2 in ischemia and status epilepticus.
The functionally critical arginine R residue within TM2 is not encoded by the GluR2 gene, but rather arises
by adenosine-specific RNA editing of the double-stranded
2. The AMPAR gene family pre-mRNA, which converts an adenosine residue in the
glutamine codon to an inosine [50,136] Fig. 1. RNA AMPARs mediate fast excitatory synaptic transmission
editing, the process by which genomically encoded in- in the vertebrate central nervous system. AMPARs are
formation is enzymatically modified, is an important mode assemblies of four subunits, GluR1-4 or GluRA-D,
of receptor regulation [8,78,97]. To date, three structurally- encoded by separate genes which are differentially ex-
related RNA-editing enzymes with adenosine deaminase pressed throughout the CNS for review, see [53,130].
activity have been identified in mammalian tissues: AMPARs assembled from combinations of GluR1, GluR3
ADAR1 dsRNA-specific adenosine deaminase, dsRAD or and or GluR4 subunits lacking the GluR2 subunit are
DRADA [63,74,98,107]; ADAR2 dsRNA-specific editase
21 21
permeable to Ca and Zn
and have doubly rectifying 1, RED-1 [43,69,82,85,97]; and RED2 dsRNA-specific
current–voltage relations due to voltage-dependent block editase 2 [82]. Each of the editing enzymes contains two
by intracellular polyamines [21,52,152]. The presence of to three dsRNA binding domains within the amino-termi-
21
edited GluR2 subunit greatly reduces Ca permeability
nal domain and deaminase activity within the carboxy-
192 H
Table 1
a
Studies supporting the GluR2 hypothesis Condition
Cell type Refs
Suppression of GluR2 gene expression Developing brain
various [108,111]
Transient global ischemia CA1 pyramidal cells
[3,44,49,109–111,113] Oxygen–glucose
dissociated hippocampal [159]
deprivation neurons
Status epilepticus CA3 pyramidal cells
[36–38,45,70,71,111,113,114] Mutant spastic rats
cerebellar Purkinje and [79]
granule cells Schizophrenia
parahippocampal [34]
pyramidal cells Amyotrophic lateral
spinal motor neurons [153]
sclerosis Alzheimer’s disease
entorhinal cortex [144]
GluR2 knockdown-induced neuronal death Adult rat and gerbil
hippocampal pyramidal [100]
neurons Developing brain
hippocampal pyramidal [39]
neurons
21
Expression of Ca -permeable AMPARs
Developing brain retinal ganglion cells
[120,139] Transient global ischemia
CA1 pyramidal cells [44,146,147]
Oxygen–glucose deprivation dissociated hippocampal
[159] neurons
Editing-deficient GluR2 mice CA3 pyramidal cells
[16]
21
Neurotoxicity mediated by Ca -permeable
AMPARs Primary cultures
cerebellar Purkinje cells [15]
Primary cultures neocortical neurons
[77,151,156] primary cultures
spinal motor neurons [5]
cell line oligodendroglial lineage
Oxygen–glucose dissociated hippocampal
[159] deprivation
neurons Editing-deficient GluR2 mice
CA3 pyramidal cells [16]
a
Modified from [109].
Fig. 1. Proposed secondary structure of GluR subunits depicting critical sites conferring functional diversity on AMPA receptors. The figure on the left is a schematic representation of a GluR-receptor subunit in its linear sequence and inserted into the cell membrane. Features of the predicted GluR-protein
structure include: 1 a large extracellular N-terminus domain; 2 three transmembrane-spanning domains TM1, TM3 and TM4; 3 a fourth hydrophobic segment M2 that is thought to make a hairpin turn within the membrane and line the ion channel [158]; 4 two extracellular segments S1
and S2 that are predicted to form the binding domains for agonists [138]; and 5 an intracellular C-terminus domain. The position of the alternatively spliced flip flop module and those of edited residues are shown. The alternative amino acids of editing sites are indicated using the single-letter code. The
Table lists the average percentages of unedited and edited GluR2 subunits as evaluated by PCR analysis and adult rat RNA. Data for the Q R site are from [20] and those for the R G site from [76]. Reprinted from [109].
H . Tanaka et al. Brain Research 886 2000 190 –207
193
terminal domain [6]. Whereas ADAR1 and ADAR2 are at the R G site and splicing of the flip flop cassettes of
expressed in most mammalian tissue; RED2 is expressed GluR1-4 are developmentally regulated. During early
exclusively in the brain [63,82,97]. postnatal life, mammalian neurons express only GluR flip
ADAR2 is an extremely efficient and specific double- splice forms; expression of GluR flop forms occurs at a
stranded RNA editase. In neurons of mammalian brain, later stage of development, leading to a reduction in the
ADAR2 edits primary transcripts encoding glutamate steady-state phase of glutamate-evoked currents [87,88].
receptor subunits at the ‘Q R’ site, thereby altering the gating and ionic permeability properties of the transmitter-
2.3. Transcriptional regulation of GluR2 activated channel [83,124,131]. The other main target of
ADAR2 is the serotonin 5HT-2C receptor, a member of Recent studies indicate the presence of a functional
the large superfamily of G protein-coupled receptors [23]. repressor element 1 RE1-like silencer in the proximal
ADAR2 edits the 5HT-2C pre-mRNA at a site near its 59 promoter of the GluR2 gene [92] and show that under
end to convert a genomically-encoded isoleucine residue to physiological conditions, the GluR2 gene is under tran-
valine. Fully edited versions of the serotonin 5HT-2C scriptional control by REST RE1 element specific tran-
receptor couple with less efficiency to G proteins. scription factor, also known as NRSF or XBR, a gene
In neonatal and adult rat brain, virtually 100 of GluR2 silencing factor which renders it highly neuron selective
mRNA is edited at the Q R site to yield GluR2 subunits [56,92] Fig. 2. Recombinant REST represses transcrip-
21
that form Ca -impermeable AMPARs. The unedited form
tion of the GluR2 gene by recruiting the co-repressors is detectable only in prenatal life E14 to P0 but never
Sin3A and histone deacetylase to the RE1 site of the exceeds 1 of total GluR2 mRNA [21]. In human brain
GluR2 promoter [56]. [96], Q R editing is somewhat less in substantia nigra
REST, a gene silencing factor which binds the RE1 72, striatum 89 and fetal tissue 96, suggesting
regulatory element, is thought to serve a critical role in that editing efficiency may vary among species and or
differentiation by repression of a subset of neuron-specific developmental stages.
genes Fig. 2. Known target genes including NaCh II In the AMPAR family, desensitization kinetics are
[17,51], calbindin I [1], synaptotagmin IV [10], neuronal controlled by editing of GluR2 through GluR4 at a site
nicotinic ACh receptor a [154] and AMPAR subunit
2
preceding the putative fourth transmembrane region [75]. GluR2 [56,90]. REST, a member of the Kruppel family of
Editing at the ‘R G’ site is specific for GluR2, -3 and -4, zinc finger transcription factor proteins, contains two
and is about 80–90 complete in adult rat brain. The repressor motifs within the N- and C-terminal domains and
degree of RNA editing at the R G site increases with age nine zinc finger motifs contained within the central
through the embryonic and postnatal periods in a subunit- portion of the protein. Molecular diversity of REST is
and splice variant-specific manner [75]. conferred by alternative RNA splicing which generates at
least six splice variants, many of which are truncated and 2.2. GluR2 RNA splicing
exhibit reduced trans-repressor activity and DNA binding capacity [104]. The structure of the REST gene and
In addition to RNA editing, further molecular diversity regulation by alternative RNA splicing are conserved in
of AMPAR subunits is conferred by alternative RNA human, mouse and rat.
splicing of GluR1-4. AMPAR subunits exist as either of In early embryogenesis, REST is expressed at high
two distinct isoforms termed ‘flip’ and ‘flop’, which are levels specifically in non-neural tissues and in undifferen-
generated by alternative splicing of a 114 bp region tiated neural precursor cells [30,127,129] and is thought to
immediately adjacent to the R G editing site. Alternative serve a critical role by repression of a subset of neuron-
splicing introduces one of two functionally critical casset- specific genes [30,127,128]. The pattern of REST expres-
tes of 38 amino acids flip or flop [135] into the sion in the immature nervous system is inversely correlated
extracellular loop of the GluR subunit. RNA editing at the with patterns of known target genes. In adult rat brain
R G site and splicing at the flip flop site act cooperatively under physiological conditions, REST is expressed by
to control the desensitization and recovery rates of neuronal cells, but less abundantly than in non-neuronal
AMPAR responses
[131]. The
allosteric modulator
cells. Highest levels of REST expression occur in neurons cyclothiazide strongly attenuates desensitization in flip but
of the hippocampus, pons medulla and midbrain. Although not in flop splice variants of recombinant AMPARs [105].
all splice forms are expressed in neuronal cells, two are Consistent with their extracellular position, neither the
neuron-specific. These splice forms contain short neuron-
21
R G site nor the flip flop cassettes affects Ca per-
specific exon N cassettes, are highly truncated and meability of AMPAR channels.
exhibit greatly reduced DNA binding activity. The finding As flip and flop splice forms of AMPARs differ in their
of neuron-specific REST transcripts in adult brain suggests functional properties, their differential spatio-temporal
that REST plays a critical role in maintenance of neuronal expression within the hippocampus would be expected to
identity by modulating expression of specific genes in have important physiological consequences. RNA editing
mature adult neurons.
194 H
Fig. 2. Models of neuron restrictive silencer element NRSE, RE-1-silencing trancription factor REST, REST function and GluR2 promoter. A DNA sequence of the neuron restrictive silencer element NRSE consensus, which serves as a recognition sequence for REST. B Schematic representation of
the repressor element 1RE-1-silencing transcription factor REST protein. C Function of NRSF REST in non-neuronal cells and neuronal cells. D Schematic showing the location of the silencer in the GluR2 promoter showing silencer element. A,B, reprinted from [61]; C, reprinted from [127]; D,
reprinted from [92].
Under pathological conditions, REST expression is mRNAs encoding them are localized to the spines of
markedly upregulated in hippocampal neurons. Kainic hippocampal neurons. A number of proteins implicated in
acid-induced status epilepticus induces a dramatic, but synaptic plasticity and the mRNAs encoding them are
transient upregulation [56] of REST in dentate granule localized to spines of hippocampal neurons. These include
cells and a pronounced, long-term 24 h or longer microtubule-associated protein 2 MAP2 [40,65], the a
21
upregulation of REST in hippocampal pyramidal neurons subunit of Ca
calmodulin-dependent protein kinase IIa following severe limbic seizures [104]. These observations
CamKIIa [19], the activity-related cytokseletal protein raise the possibility that other neuronal insults as, for
ArC [19], the RNA binding protein, CREB [31], RNA example, global ischemia, also induce REST expression in
polymerase III transcript BC1 [145], and AMPAR and neurons destined to die.
NMDAR mRNAs [41,86] for review, see [7]. Thus, translational regulation of specific transcripts within de-
2.4. Translational regulation of the GluR2 transcript ndrites would be expected to provide fine control over the
temporal and spatial extent of gene expression, including Recent interest in translational control of synaptic
those for glutamate receptor subunits. proteins has been heightened by the observation that a
Translational efficiency is under the control of two number of proteins implicated in synaptic plasticity and the
classes of RNA binding proteins, 1 translational factors,
H . Tanaka et al. Brain Research 886 2000 190 –207
195
which regulate the initiation and elongation of mature this mechanism comes from the finding of pools of
transcripts, and 2 translational repressor and enhancer AMPARs within spines [118], the high concentration of
factors. In addition, translational efficiency is influenced by NSF in the hippocampus [54,116], its high localization
transcript-specific structural motifs, which serve as recog- within hippocampal PSDs [155], and its accumulation in
nition sequences for the RNA binding proteins. These PSDs following transient cerebral ischemia [55].
include: 1 sequences residing in the 59 and 39 UTR; 2 alternate or additional 59-UTR AUG codons or their
cognate short open reading frames; 3 consensus se-
4. Regulation of GluR2 expression in
