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The Journal of Neuroscience, February 15, 1998, 18(4):1383-1392
Differential Regional Expression and Ultrastructural Localization
of  -Actinin-2, a Putative NMDA Receptor-Anchoring Protein, in
Rat Brain
Michael
Wyszynski1,
Viktor
Kharazia2,
Roopal
Shanghvi1,
Anuradha
Rao3,
Alan H.
Beggs4,
Ann Marie
Craig3,
Richard
Weinberg2, and
Morgan
Sheng1
1 Howard Hughes Medical Institute and Department of
Neurobiology, Massachusetts General Hospital and Harvard Medical
School, Boston, Massachusetts 02114, 2 Department of Cell
Biology and Anatomy, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599, 3 Department of Cell and
Structural Biology, University of Illinois, Urbana-Champaign, Illinois
61801, and 4 Genetics Division, Children's Hospital and
Harvard Medical School, Boston, Massachusetts 02115
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ABSTRACT |
Fast chemical neurotransmission is dependent on ionotropic
receptors that are concentrated and immobilized at specific
postsynaptic sites. The mechanisms of receptor clustering and anchoring
in neuronal synapses are poorly understood but presumably involve molecular linkage of membrane receptor proteins to the postsynaptic cytoskeleton. Recently the actin-binding protein  -actinin-2 was shown to bind directly to the NMDA receptor subunits NR1 and NR2B (Wyszynski et al., 1997 ), suggesting that -actinin-2 may function to
attach NMDA receptors to the actin cytoskeleton. Here we show that
-actinin-2 is localized specifically in glutamatergic synapses in
cultured hippocampal neurons. By immunogold electron microscopy, -actinin-2 is concentrated over the postsynaptic density (PSD) of
numerous asymmetric synapses where it colocalizes with NR1 immunoreactivity. Thus -actinin-2 is appropriately positioned at the
ultrastructural level to function as a postsynaptic-anchoring protein
for NMDA receptors. -Actinin-2 is not, however, exclusively found at
the PSD; immunogold labeling was also associated with filaments and the
spine apparatus of dendritic spines and with microtubules in dendritic
shafts. -Actinin-2 showed marked differential regional expression in
rat brain. For instance, the protein is expressed at much higher levels
in dentate gyrus than in area CA1 of the hippocampus. This differential
regional expression implies that glutamatergic synapses in various
parts of the brain differ with respect to their -actinin-2 content
and thus, potentially, the extent of possible interaction between
-actinin-2 and the NMDA receptor.
Key words:
-actinin; NMDA receptor; GKAP; glutamatergic synapses; postsynaptic density; actin cytoskeleton
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INTRODUCTION |
The NMDA receptor subclass of
ionotropic glutamate receptors has been implicated in the mechanisms of
synaptogenesis, synaptic plasticity, and excitotoxicity
(Constantine-Paton et al., 1990; Bliss and Collingridge, 1993; Choi,
1995). Like other ionotropic glutamate receptors, NMDA receptors are
concentrated at postsynaptic sites in glutamatergic synapses. The
molecular mechanisms by which neurotransmitter receptors are clustered
and immobilized at synaptic locations is an active area of research
that promises to reveal new insights into the structural and functional
organization of synapses.
NMDA receptor activity in neurons exhibits mechanosensitivity (Paoletti
and Ascher, 1994) and is dependent on the integrity of filamentous
actin (F-actin) (Rosenmund and Westbrook, 1993), suggesting a
functionally important interaction between NMDA receptors and the
postsynaptic actin cytoskeleton. A number of intracellular proteins may
link NMDA receptors to the cytoskeleton. For instance, the NMDA
receptor subunit NR2 binds to the postsynaptic density (PSD)-95 family
of putative ion channel-clustering proteins (Kornau et al., 1995;
Niethammer et al., 1996; Sheng and Kim, 1996), but whether this
interaction links NMDA receptors to the cytoskeleton is unclear. More
recently, -actinin-2, a member of the spectrin/dystrophin family of
actin-binding proteins, was shown to bind directly to the NMDA receptor
subunits NR1 and NR2B using yeast two-hybrid and in vitro
binding assays (Wyszynski et al., 1997). Given that -actinins are
actin-binding proteins, we hypothesized that -actinin-2 might
function as a bridging protein between NMDA receptors and actin at
postsynaptic sites. Some circumstantial evidence of this has been
obtained in neurons. At the light microscopic level in cultured
hippocampal neurons, -actinin-2 shows punctate colocalization with
NMDA receptors at presumptive synaptic sites (Wyszynski et al., 1997).
In addition, -actinin-2 can be coimmunoprecipitated in a complex
with NMDA receptors from rat brain (Wyszynski et al., 1997). However,
the subcellular localization of -actinin-2 has not been studied in
detail in vivo. The present study tests this hypothesis by
asking whether -actinin-2 is specifically concentrated in the PSD of
glutamatergic synapses in rat brain and by investigating whether
-actinin-2 colocalizes with the NMDA receptor at the ultrastructural
level. We also provide here the first report on the developmental and
spatial patterns of -actinin-2 expression in rat brain.
Interestingly, -actinin-2 protein exhibits marked differential
regional expression in rat brain, implying molecular heterogeneity of
glutamatergic synapses in different parts of the CNS.
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MATERIALS AND METHODS |
Antibodies, expression constructs, and
immunoblotting. Antibodies against -actinin-2, NR2B, and PSD-95
have been described (Sheng et al., 1994; Kim et al., 1995; Wyszynski et
al., 1997). Monoclonal anti-Ca2+/
calmodulin-dependent protein kinase II antibody (Chemicon, Temecula, CA) and anti-NR1 monoclonal antibodies 54.1 (PharMingen, San Diego, CA)
were used for immunoblotting at 1 µg/ml. -Actinin-2 and
-actinin-3 expression constructs were prepared by subcloning the
full-length cDNAs for -actinin-2 or -actinin-3 into the
EcoRI site of the mammalian expression vector pcDNA3
(Invitrogen, San Diego, CA). COS-7 cells were transfected using the
lipofectamine method (Life Technologies, Grand Island, NY). Preparation
and immunoblotting of COS-7 cell lysates and brain membranes were
performed as described (Kim et al., 1997). All proteins were visualized
using peroxidase-conjugated secondary antibodies and enhanced
chemiluminescence (ECL) (Amersham, Arlington Heights, IL).
Immunohistochemistry on brain and cultured neurons.
Immunohistochemistry on floating 50 µm brain sections was performed
as described (Kim et al., 1996) and was visualized using the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB) or using Cy3- or FITC-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Hippocampal neuronal cultures were
prepared as described (Goslin and Banker, 1991). Primary antibodies
were used at the following concentrations: 4B2 (1 µg/ml), EA-53
(1:20,000 dilution; Sigma, St Louis, MO), anti-GAD monoclonal antibodies (1 µg/ml; Boehringer Mannheim, Indianapolis, IN), and anti-GAD-6 monoclonal antibodies (1:2 dilution; Developmental Studies
Hybridoma Bank, University of Iowa, Iowa City, IA); antibodies were
visualized with FITC- and Cy3-conjugated secondary antibodies (2.5 µg/ml; Jackson ImmunoResearch) for rat brain sections and with FITC-
and Texas Red-conjugated secondary antibodies (1 µg/ml; Vector
Laboratories) for cultured hippocampal neurons. DAB brain sections and
cultured hippocampal neurons were viewed using a Zeiss Axioskop
microscope; fluorescent brain images were recorded using a BioRad MRC
1000 confocal microscope.
Electron microscopy. Male Sprague Dawley rats (200-350 gm)
were anesthetized with pentobarbital (60 mg/kg), briefly flushed with
heparinized saline, and perfused intra-aortically with 0.1 M phosphate buffer, pH 7.4 (PB), containing 4% freshly
depolymerized paraformaldehyde and either 0.1% glutaraldehyde (for
immunoperoxidase labeling) or 2% paraformaldehyde and 2%
glutaraldehyde (for immunogold labeling). Forty-micrometer-thick
frontal sections from brain were cut using a vibratome and collected in
PB.
Pre-embedding immunoperoxidase staining for electron microscopy was
performed using mouse monoclonal -actinin antibody EA-53. Sections
were pretreated sequentially in 1% sodium borohydride, 50% ethanol,
and 3% hydrogen peroxide in PBS and then blocked in 10% normal donkey
serum. Sections were then incubated sequentially in EA-53 (diluted
1:50,000-80,000 in PBS containing 0.01% Triton X-100) overnight,
biotinylated donkey anti-mouse serum (1:250 dilution; Jackson
ImmunoResearch) for 2 hr, and ExtraAvidin-peroxidase complex (1:5,000
dilution; Sigma) for 1 hr. After revealing the peroxidase with
nickel-enhanced diaminobenzidine, sections were osmicated, stained en
bloc in uranyl acetate, and wafer-embedded in Epon-Spurr resin or, for
combining with postembedding immunogold labeling, were processed
without osmium (as described below). To stabilize immunoperoxidase in
the absence of osmium, we incubated sections in platinum chloride
(0.5% in 0.1 M maleate buffer) before embedding.
For postembedding immunogold, sections were embedded as described by
Phend et al. (1995). Briefly, sections were treated sequentially over
ice in 1% tannic acid in 0.1 M maleate buffer, pH 6.0; 1% uranyl acetate; 0.5% iridium tetrabromide (Pfaltz and Bauer,
Waterbury, CT); 50 and 70% ethanol; 1% phenylenediamine hydrochloride
in 70% ethanol; and 1% uranyl acetate in 70% ethanol and then
dehydrated in 80, 95, and 100% ethanol. Sections were then immersed in
propylene oxide and infiltrated with Epon-Spurr resin. After overnight
infiltration in resin, sections were sandwiched between strips of Aclar
plastic film, flattened between microscope slides, and polymerized at 60°C for 36 hr. Chips from layers II-III of S1 cortex were glued onto plastic blocks. Thin sections (~100 nm) were cut, collected on
300-mesh uncoated nickel grids, and treated with Quick-Coat (Kiyota
Express, Elk Grove, IL) for improved section adhesion.
For single-labeling, polyclonal -actinin-2 specific antibodies 4B2
(Wyszynski et al., 1997) were used as described (Phend et al., 1995).
Briefly, grids were washed with Tris-buffered saline containing 0.005%
Tergitol NP-10 (TBS/T) at pH 7.6, incubated at 37°C overnight in
TBS/T containing a 1:8,000-10,000 dilution of 4B2, rinsed in TBS/T at
pH 7.6, transferred to TBS/T at pH 8.2, and incubated in TBS/T
containing a 1:20 dilution of secondary gold-conjugated antibody
IgG-conjugated to 18 nm gold particles (Jackson ImmunoResearch) or to
F(ab) fragments conjugated to 1.4 nm gold particles (NanoProbes, Stoney
Brook, NY). The 1.4 nm particles were visualized by silver
intensification using an HQ silver kit (NanoProbes).
For the colocalization of -actinin-2 with the NMDA receptor subunit
NMDAR1 and guanylate kinase-associated protein, either postembedding
immunogold on material processed previously with pre-embedding
immunoperoxidase or postembedding immunogold with two antibodies in
adjacent thin sections was performed. Postembedding was performed as
described above using a 1:300 dilution of rabbit polyclonal anti-NMDAR1
antibodies (Petralia et al., 1994), a 1:300 dilution of
affinity-purified rabbit polyclonal anti-GKAP antibodies (Naisbitt et
al., 1997), and a 1:8000 dilution of the rabbit polyclonal -actinin-2 specific antibodies 4B2 (Wyszynski et al., 1997). Immunoperoxidase labeling was performed as described above using a
1:50,000 dilution of mouse monoclonal -actinin specific antibodies (EA-53). After immunocytochemical processing, grids were air-dried, stained with uranyl acetate and Sato's lead, and examined with a JEOL
CX200 transmission electron microscope at 80 kV. Virtually no gold
particles were seen if the primary antibody was omitted from the
procedure, and no selective labeling of spines or synaptic structures
was seen if preimmune rabbit serum was substituted for the rabbit
polyclonal antisera.
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RESULTS |
Regional and developmental expression of -actinin-2 in
rat brain
To investigate the expression of -actinin-2 protein in rat
brain, we generated anti-peptide antibodies (termed 4B2) against a
region of -actinin-2 that is not conserved in other members of the
-actinin family of proteins (Wyszynski et al., 1997). To confirm
specificity of the 4B2 antibody, we transiently transfected COS-7 cells
with either -actinin-2 or -actinin-3 cDNAs and fluorescently stained these cells with 4B2 antibodies. 4B2 immunoreactivity was
observed in COS-7 cells expressing -actinin-2 but not -actinin-3 protein (Fig. 1A). As a
control, -actinin-3 expression was confirmed in these cells using
anti-peptide antibodies (termed 5B2) that are specific for
-actinin-3 (Fig. 1A). Similarly, by
immunoblotting, 4B2 recognized -actinin-2 but not -actinin-3
protein expressed heterologously in COS-7 cells, whereas
-actinin-3-specific antibodies showed the reverse specificity of
detection (Fig. 1B). Thus 4B2 can specifically
recognize -actinin-2 by both immunostaining and immunoblotting. In
addition to 4B2, we took advantage of an independent antibody raised
against -actinin-2 and -3 (mouse monoclonal EA-53). Because
-actinin-3 is not expressed in the brain (Fig. 1B)
(A. H. Beggs, unpublished observations), EA-53 antibodies should
be specific for -actinin-2 in rat brain. On immunoblots of rat brain
membranes, both 4B2 and EA-53 antibodies react with a single band of
~100 kDa molecular weight, consistent with the known and predicted
size of -actinin-2 (Beggs et al., 1992) (Fig. 1C). The
fact that the same band is recognized by two independent antibodies is
strong evidence that this 100 kDa band represents -actinin-2
protein. 5B2 (-actinin-3) antibodies failed to detect any bands in
rat brain (Fig. 1B).
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Figure 1.
Specificity of -actinin-2 antibodies
(4B2) and -actinin-3 antibodies (5B2).
A, COS-7 cells transfected with -actinin-2 or
-actinin-3 expression constructs were stained with
4B2, which specifically recognizes -actinin-2 and not
-actinin-3, or with 5B2, which specifically reacts
with -actinin-3 and not -actinin-2. B, Western
blot analysis of COS-7 cells transfected with -actinin-2 or
-actinin-3 (Untrans, untransfected COS-7 cell
control) and of a crude synaptosomal membrane fraction from rat whole
brain (10 µg of protein) is shown. 4B2 recognizes
-actinin-2 but not -actinin-3 protein expressed heterologously in
COS-7 cells and a 100 kDa band in brain. 5B2 recognizes
-actinin-3 in COS-7 cells but detects no band in brain.
C, Crude synaptosomal membrane fraction from rat cortex
(Ctx Memb; 10 µg of protein) was separated by SDS-PAGE
and immunoblotted for -actinin-2 using EA-53 or
4B2 antibodies. Both antibodies recognize the same 100 kDa band. Positions of molecular weight markers are shown in
kilodaltons. -A-2, -Actinin-2; -A-3, -actinin-3.
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By Western blot analysis, -actinin-2 is found abundantly in crude
synaptosomal membrane fractions from neocortex, hippocampus, and
subcortical regions but at low levels in cerebellum (Fig. 2A). This regional
pattern of expression is similar to that of NR1 and of the -subunit
of Ca2+/calmodulin-dependent protein kinase II (CaMKII),
both of which are relatively concentrated in forebrain (Fig.
2A). However, in contrast to NR1, a significant
proportion of -actinin-2 fractionates into the soluble S100
supernatant fraction of brain homogenate (Fig.
2B).
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Figure 2.
Immunoblot analysis of -actinin-2 protein
expression in rat brain. A, Differential regional
expression of -actinin-2 in rat brain analyzed by immunoblotting
with 4B2 antibodies. Expression of -actinin-2
(-A-2) is high in crude synaptosomal membrane fractions from cortex (Ctx), hippocampus
(Hpc), and subcortical regions (Subcx)
but very low in cerebellum (Cbl). Equal amounts of protein (10 µg) were loaded in each lane. The regional expression patterns of NR1 and the -subunit of
Ca2+/calmodulin-dependent protein kinase II (CaMKII) are
shown for comparison. B, Presence of -actinin-2 in
membrane and soluble fractions of rat brain. Lanes were
loaded with rat brain fractions as follows: Whole br
(total brain homogenate; 20 µg of protein), Soluble
(S100 supernatant fraction of brain homogenate; 30 µg of protein),
and Memb (crude synaptosomal membrane fraction; 10 µg
of protein). Equal percentages, rather than equal mass, of membrane and
soluble fractions were loaded (the soluble fraction contained a
threefold higher concentration of total protein than did the membrane
fraction). Filters were probed with -actinin-2 4B2 antibodies or
with NR1 antibodies. Unlike NR1 protein, a significant amount of
-actinin-2 protein from brain is present in the soluble fraction.
C, Expression of -actinin-2 protein during postnatal cortical development compared with the expression of PSD-95, NR2B, and
NR1 proteins. An equal mass (20 µg of protein) of cortical membranes
from various ages were immunoblotted for -actinin-2. The same
immunoblot filter was stripped and reprobed with the PSD-95, NR2B, and
NR1 antibodies. -Actinin-2 protein expression increases to adult
levels during the first 2 weeks of postnatal development, as do PSD-95
and NR1 expressions. Numbers indicate the postnatal age
of rats in days. Positions of molecular weight markers are shown in
kilodaltons.
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During postnatal development, -actinin-2 protein is already
detectable in neonatal rat cerebral cortex [age, postnatal day 1 (P1)] but increases during the next 2 weeks to reach high levels of
expression by ~2 weeks after birth (Fig. 2C). The temporal pattern of -actinin-2 expression is similar to that of NR1 and PSD-95 (Fig. 2C), two proteins of the postsynaptic density
that are complexed with -actinin-2 in vivo (Wyszynski et
al., 1997). In contrast, NR2B (a modulatory NMDA receptor subunit that
also binds directly to -actinin-2) is present at approximately equal amounts throughout the postnatal period (Fig. 2C). The
increase in NR1 and PSD-95 expression and the stable expression of NR2B are in agreement with previous findings (Cho et al., 1992; Sheng et
al., 1994). Thus during the first 2 weeks of postnatal development, there is a parallel induction in the expression of -actinin-2, NR1,
and PSD-95 proteins, coincident with an active period of synaptogenesis
in the cortex.
Immunohistochemical localization of -actinin-2 in rat brain
The previous study of -actinin-2 focused on immunolocalization
in cultured hippocampal neurons (Wyszynski et al., 1997). In this study
EA-53 and 4B2 antibodies were used for immunohistochemical localization
of -actinin-2 at the cellular and subcellular levels in adult rat
brain. These independent antibodies gave essentially identical staining
patterns, suggesting that they recognize the true distribution of
-actinin-2 protein. Results using EA-53 are primarily shown here
because this monoclonal antibody gave a higher signal-to-noise
ratio.
Light microscopic immunocytochemistry reveals that -actinin-2
protein is differentially expressed in different regions of the brain
(Fig. 3). Staining is prominent in
forebrain structures, particularly in the striatum, hippocampus, and
cortex (Fig. 3A). In the neocortex, -actinin-2 staining
shows layer specificity, being strongest in layers II/III and V and
weak in layer IV (Fig. 3B). At the cellular level in cortex,
expression of -actinin-2 is prominent in pyramidal neurons and in
scattered interneurons. Immunoreactivity is prominent in the dendritic
processes of pyramidal neurons and relatively absent from their cell
bodies, which stand out as "holes" against the neuropil staining
(see Fig. 3G). -Actinin-2 immunoreactivity is strikingly
punctate in character both on pyramidal cell dendrites (Fig.
3C) and in the neuropil of the neocortex (best demonstrated
by immunofluorescence confocal microscopy; Fig. 3G). The
pattern of punctate -actinin-2 staining along apical dendrites
suggests localization in dendritic spines (Fig. 3C). In
contrast, scattered interneurons in cerebral cortex are intensely immunoreactive for -actinin-2 in their somata as well as in their dendritic processes (Fig.
3B,E,F).
Somatodendritic -actinin-2 immunostaining is also prominent in cells
of the striatum (Fig. 3H). Diencephalic staining is
seen in the thalamic reticular nucleus and lateral hypothalamic nuclei
(data not shown). In the midbrain, staining is prominent in substantia
nigra (Fig. 3A). In the cerebellum, -actinin-2
immunostaining is very weak (Fig. 3I), consistent with the low level indicated by immunoblotting (Fig.
2A). In general, white matter and glial cells showed
no -actinin-2 labeling.
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Figure 3.
Immunohistochemical localization of
-actinin-2 protein in adult rat brain.
A-I, Sagittal (A,
B, H, I) and
coronal (C-G) sections labeled with
-actinin-2 antibody EA-53 and visualized via immunoperoxidase (A-F, H,
I) and immunofluorescence
(G) labeling. A, Low resolution micrograph of a rat brain sagittal section showing -actinin-2 staining particularly in the striatum (St), cortex
(Ctx), hippocampus (Hpc), and substantia
nigra (Sn). B, Layer-specific expression in cerebral cortex with predominent -actinin-2 staining in layers II/III and V. Note the -actinin-2-stained apical dendrites emanating from layer V pyramidal neurons and the low levels of staining in layer
IV neuropil. Dark dots in the micrograph represent
intensely labeled cell bodies of a population of interneurons.
C, High-magnification view of -actinin-2
immunoreactive puncta decorating apical dendrites of layer V pyramidal
neurons. D, Cortical layer V showing -actinin-2 immunoreactive processes (arrows) extending a short
distance from the base of pyramidal cell bodies
(arrowheads). E, F,
Intense -actinin-2 staining in scattered cortical interneurons
showing diffuse (E) and punctate
(F) somatodendritic staining patterns. G, High resolution fluorescence confocal micrograph of
cortical layer V showing punctate nature of -actinin-2
immunoreactivity in neuropil. Cell bodies (arrowheads)
are relatively spared of -actinin-2 staining. H,
Dense -actinin-2 immunostaining in cells of the striatum.
I, -Actinin-2 immunostaining in the cerebellum that
is very weak or undetectable. m, Molecular layer;
p, Purkinje cell body layer; g, granular
layer; w, white matter. Scale bars: A,
2.5 mm; B, H, 300 µm; I,
150 µm; C, D, G, 50 µm; E, F, 30 µm.
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In the hippocampal formation, -actinin-2 protein is expressed
throughout the dendritic fields of all regions (Fig.
4A), but staining is
strongest in the molecular layer of dentate gyrus and in area CA2
(Figs. 3A, 4A). As observed in neocortex,
the cell bodies of hippocampal pyramidal neurons are relatively
unstained, whereas the dendrites of these neurons show dense
-actinin-2 immunoreactivity that is remarkably punctate when viewed
by high resolution confocal microscopy (Fig. 4C). As seen in
the cerebral cortex, scattered interneurons in the hippocampal region
are heavily labeled for -actinin-2 (data not shown).
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Figure 4.
Differential regional expression of -actinin-2
protein in the hippocampal formation. A, DAB
histochemistry showing -actinin-2 immunostaining pattern in
hippocampus. -Actinin-2 protein is expressed throughout the
dendritic fields of all regions, but staining is strongest in the
molecular layer of dentate gyrus and in area CA2. The negative image of
the photomicrograph is shown to improve contrast. B,
Fluorescence immunocytochemistry showing the dashed line
pattern of -actinin-2 immunostaining seen in hippocampus,
interspersed among the cell bodies of the pyramidal cell layer in CA1.
C, High resolution confocal image of -actinin-2
immunostaining showing bright puncta on the cell bodies and apical
dendrites of CA3 pyramidal cells. Bright puncta are also present on
basal dendrites of CA3 pyramidal cells (data not shown).
DG, Dentate gyrus; m, molecular layer;
p, stratum pyramidale; sl, stratum
lucidum; so, stratum oriens; sr, stratum radiatum. Scale bars: A, 500 µm; B, 50 µm; C, 30 µm.
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In addition to the punctate dendritic and neuropil staining found in
both neocortex and hippocampus, we reproducibly observed a curious
pattern of bright -actinin-2 immunoreactivity extending a short
distance from the base of pyramidal neurons. Best seen in layer V
pyramidal neurons, it often resembled a short dashed line, perhaps corresponding to the path of the initial axon
segment of these neurons (see Figs. 3D, 5E).
Alternatively, it may reflect intradendritic staining (A. Rao and
A. M. Craig, unpublished observations). A similar bright
dashed line pattern of -actinin-2 staining could be seen
in the hippocampus, interspersed among the cell bodies of the pyramidal
and granule cell layers (Fig. 4B). This pattern of
-actinin-2 staining was noted with both EA-53 and 4B2
antibodies.
Selective localization of -actinin-2 at
glutamatergic synapses
-Actinin-2 binds directly to NMDA receptors, and its
immunocytochemical distribution overlaps with NMDA receptors at
synaptic sites in cultured neurons (Wyszynski et al., 1997). The highly punctate pattern of -actinin-2 staining on pyramidal cell dendrites in rat brain is consistent with its localization at dendritic spines
(Figs. 3C,G, 4C). An important
question raised by these findings is whether -actinin-2 is present
specifically in synapses containing NMDA receptors (that is,
glutamatergic synapses) or whether it is a general postsynaptic protein
present also in other kinds of synapses such as GABAergic synapses.
This question is best addressed in cultured neurons, in which
individual synapses are easier to resolve than in brain sections.
In primary cultures of hippocampal neurons, -actinin-2
staining was present in a punctate pattern often corresponding to dendritic spines (Fig. 5A).
The spiny -actinin-2 clusters overlapped extensively with clusters
of GluR1, an AMPA-type glutamate receptor subunit (Fig.
5A-C). In contrast, -actinin-2
immunoreactivity shows no detectable overlap with that of glutamic acid
decarboxylase (GAD; Fig. 5D), a marker for GABAergic
synapses (the major type of inhibitory synapses in the brain). In some
neurons -actinin-2 is present in elongated clusters in dendritic
shafts, but these shaft clusters do not correspond to synaptic sites
(Rao and Craig, unpublished observations). The absence of
immunocolocalization between -actinin-2 and GAD is confirmed
in vivo by double labeling of the cerebral cortex (Fig.
5E-G). Thus, -actinin-2 appears to be
concentrated specifically in excitatory glutamatergic synapses, consistent with its localization in dendritic spines.
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Figure 5.
-Actinin-2 protein is specifically localized to
excitatory synapses and is absent from inhibitory synapses.
A-G, Colocalization of -actinin-2 immunoreactivity
with GluR1 in cultured hippocampal neurons
(A-C), and noncolocalization of
-actinin-2 and GAD in cultured hippocampal neurons
(D) and in cerebral cortex
(E-G). For
E-G, the cortical surface is located to
the left. -Actinin-2 is visualized by FITC secondary
antibody (green), and GluR1 and GAD are
visualized by Cy3 secondary antibody (red). Composite images (C, D, G) show
colocalization of signals as yellow.
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Ultrastructural localization of -actinin-2
The highest level of resolution in immunolocalization is
afforded by immunogold labeling viewed by electron microscopy.
Immunogold studies with 4B2 antibodies revealed -actinin-2 to be
mainly associated with the postsynaptic density and postsynaptic
membrane of numerous asymmetric synapses (Fig.
6A-E);
symmetric synapses were labeled weakly if at all (data not shown). In
dendritic spines, labeling was also commonly seen over microfilaments
and the spine apparatus (Fig. 6A-C). In
addition, labeling could be seen adjacent to microtubules in dendritic
shafts (Fig. 6F).
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Figure 6.
Immunogold labeling (18 nm gold particles)
for -actinin-2 in postsynaptic densities of asymmetric synapses.
A, B, Axospinous synapses are shown;
arrowheads point to subsynaptic labeling associated with
microfilaments in dendritic spines. C,
Arrow denotes dense labeling over the spine apparatus.
D, Arrow denotes prominent gold labeling
within the postsynaptic density. E, F,
Arrowheads show -actinin-2 labeling adjacent to
microtubules in dendritic shafts; arrow in
E points to labeling over postsynaptic density of an
axodendritic synapse; arrow in F points
to immunogold over dendrodendritic membrane apposition.
Sp, Spine; r, ribosomal complex; D1, D2, two adjacent dendrites;
asterisks mark presynaptic terminals. Scale bars, 250 nm.
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Immunoperoxidase staining was qualitatively similar to immunogold
labeling, although subsynaptic labeling was more abundant in axospinous
synapses, often completely filling the cytoplasm of spines, possibly
because of diffusion of the reaction product. By combining
pre-embedding immunoperoxidase staining for -actinin-2 with
postembedding immunogold labeling for NR1, we observed frequent colocalization (Fig. 7A); this
was most prominent in axospinous synapses. Colocalization at asymmetric
synapses was also apparent between -actinin-2 and GKAP, a
postsynaptic density protein at excitatory synapses that interacts
directly with the PSD-95/synapse-associated protein (SAP)-90 family of
putative ion channel-clustering proteins (Kim et al., 1997; Naisbitt et
al., 1997) (Fig. 7B-D). The localization of
-actinin-2 at the PSD, as revealed by immunogold EM, indicates that
-actinin-2 is appropriately positioned to interact directly with
NMDA receptors.
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Figure 7.
Colocalization of -actinin-2 with NR1
(A) and GKAP
(B-D). A, Postembedding
immunogold labeling (18 nm) for NR1 is on synapse stained also for
-actinin-2 with pre-embedding immunoperoxidase (the
star marks postsynaptic electron-dense diaminobenzidine
reaction product). B, Immunogold labeling for GKAP (12 nm gold particles) over postsynaptic density of -actinin-2-positive
profile is shown. C, D, Serial thin
sections were mounted on different grids and processed with
silver-intensified immunogold for -actinin-2
(C) or GKAP (D). One
synapse is positive for both antigens (double arrows);
the other is positive for only GKAP (arrowheads on
right). Scale bars, 250 nm; C and
D are to the same scale.
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DISCUSSION |
In our initial characterization of -actinin-2 interactions with
NMDA receptors, we provided evidence with light microscopy that
-actinin-2 is concentrated in synapses of cultured hippocampal neurons, as well as biochemical evidence that it is a core component of
PSD fractions (Wyszynski et al., 1997). In the present report, we show
that -actinin-2 is selective for glutamatergic synapses. Moreover,
using immunogold EM in the intact brain, we demonstrate that
-actinin-2 is indeed concentrated at the PSD of asymmetric synapses
and that it may be found at synapses also expressing NR1. These
ultrastructural findings indicate that -actinin-2 is close enough to
bind directly to NR1 at postsynaptic sites. Together with previous data
suggesting biochemical association of NR1 and -actinin-2 (Wyszynski
et al., 1997), these EM results strengthen the idea that -actinin-2
functions in the synapse as an anchoring protein linking NMDA receptors
to the submembrane actin cytoskeleton.
-Actinin-2 is not exclusively localized to the PSD, however.
Subsynaptic -actinin-2 labeling was found associated with
microfilaments and the spine apparatus in dendritic spines. In
addition, -actinin-2 labeling was detected in dendritic shafts
adjacent to microtubules and sometimes at sites of membrane contact
between dendrites. Some presynaptic expression of -actinin-2 can
also not be excluded given the ~10-20 nm error inherent in
immunogold localization. However, pre-embedding immunoperoxidase
labeling also supported a predominantly postsynaptic concentration of
-actinin-2. Thus -actinin-2 differs from other PSD components
[such as PSD-95 or GKAP (Naisbitt et al., 1997)] in showing
relatively widespread subcellular distribution in neurons in addition
to its accumulation at the PSD. This is not surprising given that the
-actinins are actin-binding proteins found in many cell types and at
a variety of cell junctions in non-neural cells.
The mechanism by which -actinin-2 is concentrated at synapses
remains to be determined. Two known binding partners of -actinin-2, NMDA receptors and F-actin, are concentrated in the PSD and could result in its recruitment to postsynaptic sites. We found no detectable interaction between -actinin-2 and the PSD-95 family of synaptic proteins (M. Wyszynski and M. Sheng, unpublished observations), despite
a C-terminal peptide sequence in -actinin-2 (-ETDL) that resembles
known PDZ domain-binding motifs. We speculate that the -actinin-2 C
terminal may bind to another PDZ-containing protein in synapses.
-Actinins are known actin-binding molecules, and actin is
concentrated in dendritic spines, constituting a major component of
brain postsynaptic densities (Matus et al., 1982; Hammonds, 1987; Byers
et al., 1989). Because of its ability to cross-link actin filaments,
-actinin-2 could be an important structural component of the
postsynaptic cytoskeleton. Interestingly, Ca2+
inhibits the actin-binding activity of -actinin purified from nonmuscle tissues including brain (Duhaiman and Bamburg, 1984). Calcium
inhibition of actin cross-linking by -actinin-2 could be a possible
mechanism contributing to activity-dependent remodeling of the
postsynaptic actin cytoskeleton. In contrast to Ca2+
effects, -actinin binding to F-actin is stimulated by
PIP2 (Fukami et al., 1992), a phospholipid second messenger
the synthesis of which is regulated by RhoA, a small GTPase important
in cytoskeletal regulation. Bidirectional control of -actinin-actin
interactions via Ca2+ and PIP2 signals
could contribute to cytoskeletal remodeling during activity-dependent
structural plasticity of synapses.
The direct interaction between -actinin-2 and NMDA receptor subunits
represents an attractive mechanism for connecting NMDA receptors to
postsynaptic actin filaments. Such a link has been implied by
electrophysiological studies showing a dependence of NMDA receptor
function on the integrity of the actin cytoskeleton (Rosenmund and
Westbrook, 1993). Intriguingly, NMDA receptor anchoring to actin may be
dynamically regulated, because -actinin-2 binding to NR1 is directly
antagonized by Ca2+/calmodulin in vitro
(Wyszynski et al., 1997). Thus Ca2+ can potentially
regulate NMDA receptor attachment to actin via two mechanisms,
displacement of -actinin-2 from NR1 by calmodulin and inhibition of
the actin-binding activity of -actinin. It is possible that both of
these mechanisms could contribute to Ca2+-dependent
inactivation of NMDA receptors (Rosenmund and Westbrook, 1993; Ehlers
et al., 1996; Krupp et al., 1996). Thus -actinin-2 may play a role
in both the localization and Ca2+ modulation of NMDA
receptors.
The differential regional expression of -actinin-2 in rat brain
suggests that glutamatergic synapses in distinct parts of the brain may
differ in terms of their -actinin-2 content. This finding emphasizes
the heterogeneity of excitatory synapses with respect to molecular
composition. Whether this reflects a functional heterogeneity of
glutamatergic synapses remains to be determined. -Actinin-2 exists
at very low or undetectable levels in cerebellar granule cells,
suggesting that NMDA receptors in this region are less likely to
interact with -actinin-2. In the hippocampus, NMDA receptor function
may be different in CA1 compared with the dentate gyrus because of
differential interactions with -actinin-2. In this context, it is
intriguing that the CA1 region, the most sensitive to NMDA
receptor-mediated excitotoxicity, expresses low levels of
-actinin-2.
Outside of the brain, -actinins are concentrated at the
neuromuscular junction (Bloch and Hall, 1983), although there is no
evidence that -actinin interacts with acetylcholine receptors in
this model peripheral synapse. In addition to postsynaptic sites,
-actinins are known to accumulate at other cell junctions, most
notably at focal adhesions, the sites of contact between cultured cells
and the extracellular matrix (Clark and Brugge, 1995). In focal
adhesions, -actinin binds through its rod domain directly to the
cytoplasmic tail of  1-integrins (Otey et al., 1990), thus providing
another example of a direct -actinin interaction with an integral
membrane protein. The linkage of integrins to actin by -actinin is
thought to play a role in nucleating the integrin-directed
cytoskeletal-signaling complex at focal contacts (Clark and Brugge,
1995). Focal adhesions and synapses both represent dynamic sites of
membrane specialization where actin filaments converge, where specific
extracellular matrix contacts are made, and where signaling molecules
including tyrosine kinases are highly concentrated (Clark and Brugge,
1995). The finding that -actinin localizes to synaptic junctions
reveals that there are molecular as well as organizational similarities
between synapses and focal contacts.
Evidence converging from several lines of work is beginning to reveal
the complexity of the interactions of NMDA receptors with
cytoskeleton-associated proteins. Because NR2 subunits of NMDA
receptors bind directly to the PSD-95 family of channel-clustering molecules, the interaction between -actinin-2 and NR1 emphasizes that heteromeric membrane receptors may use multiple mechanisms to
interact with the subsynaptic cytoskeleton and with
postsynaptic-signaling molecules.
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FOOTNOTES |
Received Oct. 6, 1997; revised Dec. 1, 1997; accepted Dec. 1, 1997.
This research was supported by National Research Service Award
CA66268-02 from the National Institutes of Health (M.W.), National Institutes of Health Grants NS35050 (M.S.) and NS29879 (R.W.), Grant
AR44345 from the National Institute of Arthritis and Musculoskeletal and Skin Diseases (A.H.B.), and a grant from the Muscular Dystrophy Association (A.H.B.). M.S. is an Assistant Investigator of the Howard
Hughes Medical Institute.
Correspondence should be addressed to Dr. Morgan Sheng, Howard Hughes
Medical Institute (Wellman 423), Massachusetts General Hospital, 50 Blossom Street, Boston, MA 02114.
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Abstract
Introduction
