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Novel cyclic AMP signalling avenues in learning and memory
Ostroveanu, A
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Chapter 2
A-kinase anchoring protein 150 in the mouse
brain is concentrated in areas involved in learning
and memory
Anghelus Ostroveanu, Eddy A. Van der Zee, Amalia M. Dolga, Paul G. M. Luiten, Ulrich
L.M. Eisel, Ingrid M. Nijholt
Department of Molecular Neurobiology, Graduate School of Behavioral and Cognitive
Neurosciences, University of Groningen, The Netherlands
Brain Research, 2007, 1145(11):97-107
Chapter 2
Abstract
A-kinase anchoring proteins (AKAPs) form large macromolecular signaling complexes that
specifically target cAMP-dependent protein kinase (PKA) to unique subcellular
compartments and thus, provide high specificity to PKA signaling. For example, the
AKAP79/150 family tethers PKA, PKC and PP2B to neuronal membranes and postsynaptic
densities and plays an important role in synaptic function. Several studies suggested that
AKAP79/150 anchored PKA contributes to mechanisms associated with synaptic plasticity
and memory processes, but the precise role of AKAPs in these processes is still unknown.
In this study we established the mouse brain distribution of AKAP150 using two wellcharacterized AKAP150 antibodies. Using Western blotting and immunohistochemistry we
showed that AKAP150 is widely distributed throughout the mouse brain. The highest
AKAP150 expression levels were observed in striatum, cerebral cortex and several other
forebrain regions (e.g. olfactory tubercle), relatively high expression was found in
hippocampus and olfactory bulb and low/no expression in cerebellum, hypothalamus,
thalamus and brain stem. Although there were some minor differences in mouse AKAP150
brain distribution compared to the distribution in rat brain, our data suggested that rodents
have a characteristic AKAP150 brain distribution pattern.
In general we observed that AKAP150 is strongly expressed in mouse brain regions
involved in learning and memory. These data support its suggested role in synaptic
plasticity and memory processes.
Keywords: AKAP150; localization; immunohistochemistry; cAMP-dependent protein
kinase
40
AKAP150 in the mouse brain
Introduction
cAMP-dependent protein kinase (PKA) is involved in several intracellular signaling
cascades and it regulates multiple cellular functions (Scott, 1991; Skalhegg & Tasken,
2000). A potential mechanism to explain how such a multifunctional and broad substrate
kinase mediates precise signaling events, is colocalization of its substrate to specific
subcellular compartments. Compartmentalization arises in part from the association of the
enzyme with so-called A-kinase anchoring proteins (AKAPs) (Glantz et al., 1993;
Lohmann, et al., 1984). AKAPs represent a group of more than 70 identified functionally
related proteins (Wong & Scott, 2004). Although they share little primary structure
similarities, they all have the ability to bind PKA, and therefore to regulate specific cAMP
signaling pathways by sequestering PKA to a specific subcellular location. This
compartmentalization of individual AKAP-PKA complexes occurs through unique
targeting domains that are present on each anchoring protein.
To date, AKAPs have been identified in a wide range of species, tissues and cellular
compartments (Angelo & Rubin, 2000; Jackson & Berg, 2002, Sarkar et al., 1984; Wong &
Scott, 2004). In the mammalian brain, several AKAPs have been characterized. One of
these AKAPs is AKAP79/150. This family of proteins consists of three orthologues: bovine
AKAP75, murine AKAP150 and human AKAP79. Initially AKAP75 was identified as a
contaminant of PKA regulatory subunit II (RII) purified cytosolic brain preparations
(Bregman et al., 1991; Sarkar et al., 1984). In addition, Bregman and colleagues retrieved
AKAP150 by screening a rat cDNA library using radiolabeled RIIβ as functional probe
(Bregman et al., 1989). Finally AKAP79 was identified as a constituent of postsynaptic
densities (PSD) in human cerebral cortex (Carr et al., 1992).
Interestingly, AKAPs function as multi-assembly scaffold molecules interacting with other
signaling enzymes. AKAP79/150 has the ability to bind protein phosphatase 2B/calcineurin
(PP2B/CaN) (Dell’Acqua et al., 2002) and protein kinase C (PKC) (Faux et al., 1999)
besides PKA. By tethering both kinases and phosphatases AKAP79/150 provides a unique
platform for integrating opposite signaling events to the same subcellular site.
It has been suggested that in excitatory synapses at the PSD, AKAP79/150 targets its
anchored proteins and forms a multi protein complex with AMPA and NMDA receptors
41
Chapter 2
(AMPAR and NMDAR), synaptic adhesion molecules, and cytoskeleton proteins. These
proteins play an important role in synaptic function (Colledge et al., 2000; Kennedy, 1997;
Malenka & Bear, 2004; Yamauchi, 2002; Ziff, 1997).
The first evidence that anchoring of PKA is crucial for the regulation of synaptic function
was reported by Rosenmund et al. (1994). In their study, blocking the PKA anchoring to
AKAPs prevented the PKA-mediated regulation of AMPA/kainate currents in cultured
hippocampal neurons. Moreover, recent findings strongly indicate that anchored PKA is
crucial for maintaining AMPA currents during glutamate stimulation (Hoshi et al., 2005).
Interestingly, disruption of AKAP-PKA anchoring leads to CaN-dependent, long-term
depression (LTD)-like down-regulation of AMPAR currents, implicating an important role
for AKAP79/150 in AMPAR regulation (Tavalin et al., 2002). In general, the AKAP79/150
scaffold molecule has emerged as an important element in regulating AMPAR
phosphorylation in long-term potentiation (LTP) and LTD at the PSD (Dell’Acqua et al.,
2006; Genin et al., 2003; Snyder et al., 2005). Since strengthening or weakening of synaptic
transmission is widely considered to be the cellular mechanism that underlies learning and
memory, a role of AKAP79/150 in learning and memory can be expected.
To date, only a few immunohistochemical localization studies illustrate the distribution of
AKAP79/150 in different brain compartments in various species. In human brain high
levels of AKAP79/150 were reported at the PSD of the forebrain (Carr et al., 1992). A more
detailed analysis of AKAP150 protein distribution in the rat brain showed that AKAP150 is
widely distributed throughout the brain and is expressed in many classes of neurons that
constitute the rat CNS (Glantz et al., 1992). A more recent study focused on the distribution
of AKAP150 at rat CA1 pyramidal cell asymmetric and symmetric PSD and its
colocalization with several markers of excitatory and inhibitory receptors. In this study, the
interaction of AKAP150 with components of the excitatory PSD was confirmed, whereas
AKAP150 immunoreactivity (IR) was not associated with inhibitory synapses (Lilly et al.,
2005).
The distribution of AKAP150 protein in the mouse brain has not been established yet. To
elucidate the specific role of AKAP150 in learning and memory processes it may be
important to use genetically modified mice in future research. Therefore, besides
characterizing AKAP150 expression throughout the whole mouse brain, we specifically
42
AKAP150 in the mouse brain
focused on AKAP150 expression in areas known to be involved in learning and memory
processes. We established the distribution of AKAP150 protein in the mouse brain using
immunohistochemistry and Western blot techniques.
Material and Methods
Animals and housing conditions
The present study is based on brain tissue from 9-week-old male C57Bl/6J inbred mice
(n=8) obtained from Harlan, Horst, The Netherlands. Animals were group-housed in
standard macrolon cages placed in an environmentally controlled room for temperature (22
± 1 °C) and humidity (45 ± 10%) under a 12:12h light/dark cycle with lights on at 7.00 a.m.
Animals had free access to water and standard food pellets. Collection of brain tissue was
performed after at least one week of accommodation under the above conditions. All
procedures concerning animal care and treatment were in accordance with the regulation of
the ethical committee for the use of experimental animals of the University of Groningen,
The Netherlands (License: DEC 4278A).
Western blotting
CO2/O2 anesthetized animals were quickly decapitated and brain tissue was removed on ice.
The following brain regions were excised, immediately frozen in liquid nitrogen and stored
at -80 °C before further processing: hippocampus, striatum, olfactory bulb, cortex,
cerebellum, hypothalamus and brain stem.
Brain tissue was mechanically homogenized in 10 volumes of homogenization buffer [50
mM HEPES (pH 7.4), 150 mM NaCl, 0.2 % NP-40, 4 mM EGTA, 10 mM EDTA, 15 mM
sodium pyrophosphate, 100 mM β-glycerophosphate, 50 mM sodium fluoride, 5 mM
sodium orthovanadate, 1 mM dithiothreitol, 1 mM PMSF, and Complete Mini Protease
Inhibitor Cocktail (Roche)]. The homogenate was centrifuged at 20,000 x g for 10 min at 4
°C, and the resulting supernatant was assayed for protein concentration using the Bradford
method.
43
Chapter 2
Protein samples (20 µg per sample) were separated on a 10 % SDS polyacrylamide gel and
transferred to PVDF membranes (Millipore, USA). The blots were blocked for 1 h in
blocking buffer (0.2 % I-Block (Tropix), 0.1 % Tween 20) and then incubated overnight at
4 °C either with goat anti-AKAP150 N-19 (1:500, sc-6446 Santa Cruz, CA, USA) or goat
anti-AKAP150 C-20 (1:2,500, sc-6445 Santa Cruz, CA, USA). Mouse anti-actin antibody
(1:40,000; MP Biomedicals, Irvine, CA, USA) served as control for protein loading. The
blots were incubated with alkaline phosphatase-conjugated secondary antibodies [AP
conjugated donkey anti-goat IgG (1:10,000)] (sc-2022 Santa Cruz, CA, USA) and APconjugated goat anti-mouse (1:10,000) (AC32ML, Tropix). Western blots were developed
using the chemiluminescence method (Nitroblock and CDP-Star, Tropix). The
immunoblots were digitized and quantified using a Leica DFC 320 image analysis system
(Leica, Cambridge, UK).
Immunohistochemistry
Animals were anesthetized by intraperitoneal injection with sodium pentobarbital 6%
solution and sacrificed by transcardial perfusion with saline solution containing heparin,
followed by 4 % paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB). After perfusion,
the brains were kept at 4 °C in 0.01 M phosphate buffered saline (PBS) containing 0.1%
sodium azide for 72 h. All brains were dehydrated for 48 h in a 30 % buffered sucrose
solution. After dehydration, 20 µm coronal or sagittal sections were cut on a cryostat
microtome. Sections were stored at 4 °C in 0.01 M PBS containing 0.1 % sodium azide.
The avidin/biotin immunoperoxidase staining method was used to visualize mouse
AKAP150 IR. Coronal and sagittal sections covering the whole mouse brain were rinsed in
0.01 M PBS. Sections were preincubated with 0.3 % H2O2 to reduce endogenous
peroxidase activity and afterwards rinsed in PBS. Non-specific binding sites were blocked
by preincubating the sections with 5 % normal rabbit serum in 0.01 M PBS for 30 min.
Subsequently sections were incubated either with goat anti-AKAP150 N-19 (1:500) or goat
anti-AKAP150 C-20 (1:500) in 0.01 M PBS containing 5 % normal rabbit serum and 0.3%
Triton X-100 for 2 h at room temperature (RT) and left overnight at 4 °C. Afterwards the
sections were rinsed (3 x 15 min in PBS) and incubated at RT for 2 h with biotinconjugated rabbit anti-goat antibody (1:400) (Jackson Inc.) in 0.01 M PBS containing 1 %
44
AKAP150 in the mouse brain
normal rabbit serum and 3 % Triton X-100. Another rinsing step with 0.01 M PBS at 4 °C
was followed by incubation with avidin complex containing biotinylated horseradish
peroxidase (1:400) (Vectastain ABC Kit, Burlingame, CA, USA) for 2 h at RT. Finally,
staining was visualized with 0.03 % diaminobenzidine (DAB) chromogen substrate and
0.001 % H2O2. The incubation reactions were stopped by rinsing the sections with 0.01 M
PBS.
Immunostained sections were analyzed with an Olympus BH2 microscope (Olympus,
Japan). Two independent investigators established semi-quantification of mouse AKAP150
IR. For each brain region, material from 6 animals was examined to establish AKAP150
immunostaining. The scoring system used for establishing the relative AKAP IR was
classified as follows: absent (-), low (+-), moderate (+), high (++) and very high (+++). In
addition, for a subjective quantification of AKAP150 protein distribution in the mouse
brain, relative densitometry measures for some major brain regions and nuclei have been
determined (Table 2) (Quantimet 550 IW, Cambridge, UK). Photographs were taken with a
DM1000/DFC280 Leica image analysis system (Leica, Cambridge, UK).
Antibody specificity
Both AKAP150 antibodies are affinity purified goat polyclonal antibodies raised against a
peptide mapping at the carboxyl terminus part (C-20) or N-terminus (N-19) of AKAP150 of
rat origin. They showed, in parallel staining experiments, high specific AKAP150 IR in the
mouse brain and displayed a similar pattern of staining. Because polyclonal antibodies were
used, different affinities and avidities of the antibodies could influence the staining
intensity. Therefore Table 1 reflects only relative amounts of AKAP150 IR, rather than a
quantitative comparison between the two AKAP150 antibodies. The specificity of both
antibodies in the mouse brain was assessed by parallel staining performed without primary
antibodies and with secondary antibodies alone and showed no staining signals (data not
shown). In addition, incubation of the conjugate with AKAP150 antibody/blocking peptide
(1:10; Santa Cruz, CA, USA) was carried out at 4 °C overnight, followed by 25 min
centrifugation (12,000 rpm), before application to the tissue sections. Following
preabsorption, no AKAP150 staining was observed (Fig. 4B). Furthermore, preincubation
45
Chapter 2
steps were performed to increase the specificity of AKAP150 antibodies in the mouse
brain.
Results
Western blot analysis of AKAP150 expression levels in various brain compartments
Western blot analysis with two AKAP150 antibodies (a N-terminal or C-terminal antibody)
in protein extracts from different mouse brain regions revealed the highest expression level
of AKAP150 protein in cortex and striatum (Fig. 1). High expression levels were also
detected in the hippocampus and olfactory bulb, while the cerebellum and the
hypothalamus revealed low levels of AKAP150 expression. The lowest expression level of
A
AKAP150 expression level (%)
AKAP150 was found in the brain stem (Fig. 1).
140
120
100
80
60
Fig. 1 Western blot analysis of AKAP150 expression levels in
mouse brain. (A) Bar graph representing AKAP150 expression
level in different compartments of mouse brain. Values represent
mean percentages of integrated optical density (I.O.D). ± S.E.M.
Cortex was set to 100% (B) Representative Western blot with
40
protein extracts from different brain regions. Actin served as
20
control for protein load.
0
B
AKAP150
Actin
s
b s
x
ul pu um em
te u m u
or bell lam ry b am triat n st
C e ha o c S
ai
t
ct o
er
Br
C ypo lfa ipp
H
O
H
The results of the Western blot expression levels
corresponded with the distribution pattern of this
protein in the immunohistochemical analysis of the
mouse brain (Fig. 2; Tables 1 and 2).
General overview of AKAP150 IR in mouse brain
AKAP150 IR was widely distributed throughout the brain. Highest IR was found in
striatum and olfactory tubercle, but in cortex, hippocampus and amygdala AKAP150
expression was also very abundant (Fig. 2).
46
AKAP150 in the mouse brain
In several brain regions, AKAP150 IR was limited to specific cell layers (e.g. the Purkinje
cell layer of the cerebellum) or nuclei (e.g. reticular thalamic nucleus, ethmoid nucleus)
(Table 1). Some brain regions did not show any IR for AKAP150 protein (e.g. numerous
nuclei in midbrain and hindbrain) (Fig. 2; Table 1).
A
AP 1,98mm
B
C
AP 1,18mm
AP -0,70mm
CPu
CPu
LGP
LH
Acb
OT
D
AP -1,46mm
H
E
AP -2,92mm
SC
VL
BLA
AH
SNR
Fig. 2. Localization of AKAP150 protein in coronal sections of the brain. Distance from bregma (AP) is indicated
directly on the photographs (Franklin & Paxinos, 1997): (A) OT - olfactory tubercle, (B) CPu - caudate putamen,
Acb - accumbens nucleus, (C) CPu - caudate nucleus, LGP - lateral globus pallidus, LH - lateral hypothalamic
area, (D) H - hippocampus, VL - ventrolateral thalamic nucleus, BLA - basolateral amygdala, AH - anterior
hypothalamic area, (E) SC - superior colliculus, SNR - substantia nigra reticular part. Scale bar = 1 mm.
Detailed description of AKAP150 expression in mouse brain
Olfactory system
AKAP150 IR varied from moderate to relatively high in the olfactory bulb and its various
layers. Superficial layers (glomerular layer and external plexiform layer) as well as deep
layers (internal plexiform layer and granule layer) of the olfactory bulb showed moderate
expression of AKAP150 (Table 1). Only the mitral cell layer of the olfactory bulb was
highly immunoreactive for AKAP150 protein (Table 1). The olfactory tubercle showed a
different staining pattern than the rest of the olfactory system. Here we observed a very
47
Chapter 2
high AKAP150 IR pattern. The excessive staining made it difficult to distinguish
subcellular compartments within the olfactory tubercle (Figs. 2A and B).
Cerebral cortex
In mouse cerebral cortex, AKAP150 protein was found to be expressed throughout all
cortical layers (Figs. 2 and 3A). Very high AKAP150 IR levels were observed in cortical
layers I (molecular layer), II (external granular layer) and IV (internal granular layer).
Cortical layers II and IV were highly immunoreactive for AKAP150 protein in perikarya of
granule cells (Figs. 3B and C).
A
I
B
B
C
I-III
II-IV
D
C
E
V
F
VI
E
F
D
Fig. 3 Distribution of AKAP150 protein in the cerebral cortex. (A) Overview of AKAP150 protein distribution in
cerebral cortex somatosensory 1, trunk region. Boxed areas represent the localization of panel B-F. (B) Dense
staining is observed in cortical layers I-III. (C) Perikarya staining in cortical layer IV. (D) AKAP150 staining in
barrel cortex. (E) Both fibers and cell bodies are immunopositive for AKAP 150 in cortical layer V. Note the
weaker staining and less perikarya IR for AKAP150 in this layer as compared with cortical layer IV. (F)
AKAP150 IR in cortical layer VI.
In layer IV of the somatosensory region of the mouse cerebral cortex, cortical barrels
displayed a dense and characteristic AKAP150 IR pattern (Fig. 3D). Cortical layer III
(external pyramidal layer) showed pronounced AKAP150 IR (Figs. 3A and B) whereas
48
AKAP150 in the mouse brain
cortical layer V (internal pyramidal layer), which represents a principal output system of
the neocortex, revealed only moderate immunostaining for AKAP150 (Fig. 3E). The
deepest cortical layer, layer VI (polymorphic layer), was strongly AKAP150 IR. (Fig. 3F).
The highest IR of AKAP150 protein in mouse cerebral cortex was found in cerebral cortex
layer I (molecular layer) and in the stellate cells (interneurons) of both external and internal
granular layers (cortex layers II and IV). In addition, the entorhinal cortex, the major source
of afferents to the hippocampal formation, was intensely stained (Table 1).
Hippocampal formation
Although there were some differences within this laminar structure, overall the
hippocampal formation displayed high IR for AKAP150 protein (Fig. 4A; Tables 1 and 2).
A
SO
Py
C
SR
SLM
CA1
SLu
CA3
E
D
GCL
hil
C
B
ML
D
Py
E
Py
ML
GCL
hil
Fig. 4 Immunostaining pattern for AKAP150 in the hippocampal formation. (A) AKAP150 IR in stratum oriens
(SO), stratum radiatum (SR), stratum lacunosum moleculare (SLM), stratum pyramidale (Py) of CA1-3 and
stratum lucidum (SLu) of CA3 and in molecular layer (ML), dentate granular cell layer (GCL) and hilus (hil) of
the dentate gyrus. (B) AKAP150 IR after preabsorption of AKAP150 antiserum with the antigenic peptide. (C)
Higher magnification of boxed area in panel A, showing immunopositive perikarya of pyramidal neurons (Py) in
CA1, (D) Higher magnification of boxed area in panel A, showing stained pyramidal cell bodies (Py) in CA3. (E)
Higher magnification of boxed area in panel A, showing immunopositive granule cell perikarya in the dentate
gyrus (GCL).
49
Chapter 2
Especially in the CA1-3 region of the hippocampus, AKAP150 showed a very high
expression in basal dendrites (stratum oriens) and apical dendrites (stratum radiatum) of
pyramidal neurons (Fig. 4A). A somewhat lower IR was observed in both stratum
lacunosum moleculare and stratum lucidum (Fig. 4A). A characteristic staining pattern was
observed in the pyramidal cell body layer. In CA1 the pyramidal cell bodies were
moderately stained (Figs. 4A and C) whereas in CA3 pyramidal neurons perikarya were
rather densely stained (Figs. 4A and D).
The subiculum also showed high AKAP150 IR (Table 1). In the dentate gyrus, AKAP150
IR was characterized by very high staining in the granule cell dendrites of the medial and
lateral blade of the molecular layer (Fig. 4A). However, the dentate granule cells showed
only moderate staining, whereas the hilus was slightly more immunoreactive (Figs. 4A and
E). Interestingly, scattered non-principal cells in the dentate gyrus were also strongly
stained for AKAP150 protein (Fig. 4E).
Amygdala
In the amygdala, AKAP150 IR patterns revealed a high level of this protein in the various
subcompartments. The distribution of AKAP 150 was clear and strong throughout all
amygdaloid nuclei with only slight differences in the density of staining (Table 1). Uniform
but strong staining was detected in cortical amygdaloid nuclei, medial amygdaloid nucleus,
basomedial amygdaloid nucleus, amygdalohippocampal area and amygdalopiriform
transition area (Table 1, Fig. 5A).
A
Fig. 5 Distribution of AKAP150 in the
B
amygdala. (A) AKAP150 IR in the lateral
amygdaloid nucleus, dorsolateral part
LaDL
(LaDL),
CeMPV
B
amygdaloid
nucleus,
ventrolateral part (LaVL) and the anterior
part
CeL
LaVL
lateral
CeC
of
the
basolateral
amygdaloid
nucleus (BLA). (B) Higher magnification
of boxed area in panel A, showing
BLA
AKAP150 IR in the central amygdala.
The capsular part (CeC), lateral division
(CeL) and the medial posteroventral part
of the central amygdala (CeMPV) show staining in both cell bodies and fibers (see magnification in panel).
50
AKAP150 in the mouse brain
The most prominent staining in the amygdala for AKAP150 protein was detected in the
central amygdaloid nucleus, lateral amygdaloid nucleus and basolateral amygdaloid nucleus
(anterior, posterior and ventral) (Fig. 5A). The basolateral complex revealed a strong IR
whereas the central amygdala was characterized by a dense staining in which both neuronal
perikarya and fibers were detected (Figs. 5A and B; Table 1).
Septal nuclei and striatopallidal system
In these brain regions, well-defined AKAP150 IR was observed. In the septum, the lateral
nucleus was strongly stained, whereas the medial nucleus showed only faint AKAP150
staining (Fig. 2B; Tables 1 and 2).
The caudate putamen was the most strongly stained structure in the entire mouse brain. The
very dense AKAP150 IR pattern made it difficult to distinguish the subcellular distribution
of AKAP150, although some perikarya were observed. Claustrum and ventral pallidum
exhibited moderate to high AKAP150IR, while the globus pallidus showed almost no
staining (Fig. 2; Table 1).
Epithalamus and thalamus
In the epithalamus AKAP150 protein showed a moderate expression. Both the medial and
lateral habenula displayed a diffuse IR with faint perikarya staining (Table 1).
At the level of the thalamus distinct patterns of expression were detectable although the
overall distribution of AKAP150 protein in this brain region was rather low (Fig. 2; Table
1). Detailed analysis of the stained thalamus revealed moderate staining in reticular
thalamic nucleus, subgeniculate nucleus and ethmoid nucleus, where scattered fibers
(varicose fibers) were specifically stained. Low levels of AKAP150, described as faint
staining, were found in geniculate nucleus, anterior nucleus, lateral nucleus, ventral nucleus
and zona incerta (Fig. 2). The remainder of the thalamus showed no AKAP150 IR (Table
1).
Hypothalamus
In general, the expression level of AKAP150 in the hypothalamus was moderate to low.
Accordingly, the supraoptic nucleus, paraventricular nucleus and suprachiasmatic nucleus
51
Chapter 2
showed a very faint and diffuse pattern of staining (Fig. 2; Table 1). Among the regions
with the highest IR, the median eminence, arcuate nucleus and mammillary nuclei
displayed a homogeneous staining (Table 1).
Midbrain
AKAP150 protein showed a moderate to low expression in the midbrain region (Table 1).
Most of the staining was observed in the superior colliculus, substantia nigra pars reticulata
and pars compacta and periaqueductal gray (Fig. 2E). The IR in ventral tegmental area
showed only moderate levels of AKAP150 (Fig. 2E). Low levels of AKAP150 IR were
detectable in inferior colliculus and interpeducular nuclei (Table 1).
Cerebellum
In the cerebellum, AKAP150 IR showed a moderate to low expression. Most of the staining
was seen in the molecular layer of the cerebellar cortex in what appeared to be dendrites of
the Purkinje neurons (Fig. 6B). The Purkinje perikarya were not well stained although
punctuate staining of AKAP150 IR was detectable in the Purkinje layer throughout the
whole cerebellar cortex (Fig. 6A). In the granule cell layer, the staining was considered
faint, corresponding to a low expression of AKAP150 protein (Fig. 6A).
B
A
Fig. 6 AKAP150 expression in the
cerebellum. (A) AKAP150 IR in the
cerebellar molecular layer (Mol). (B)
Higher magnification of boxed area in
Gr
Mol
B
panel A, showing staining of Purkinje
neurons (arrow, also in panel A)
staining in both cell body and dendrites.
Pons and medulla oblongata
In the brainstem, the overall expression level of AKAP150 was low. Only the inferior
olivary complex and nucleus of the solitary tract showed moderate immunostaining (Table
1). The remainder of this brain structure showed low or no AKAP150 IR.
52
AKAP150 in the mouse brain
Table 1 - Expression of AKAP150 protein in various
compartments of the mouse central nervous system.
Brain region
CNterm
term
Telencephalon
Olfactory system
Glomerular layer
+
+
External plexiform layer
+
+
Mitral cell layer
++
++
Internal plexiform layer
+
+
Granule layer
+
+
Anterior olfactory nucleus
+
+
Olfactory tubercle
++
++/+++
Cerebral cortex
Layer I
+++
+++
Layer II
++
++
Layer III
++
++
Layer IV
+++
++/+++
Layer V
+
+
Layer VI
++
++
Piriform cortex
++
++
Hippocampal formation
Dentate gyrus
Granule cell layer
+
+
Molecular layer
+++
+++
Hilus
+++
++
CA1 region
Stratum radiatum
+++
+++
Stratum oriens
+++
+++
Stratum lacunosum
++
++
moleculare
Stratum pyramidale
+
+
CA3 region
Stratum oriens
+++
+++
Stratum radiatum
+++
+++
Stratum pyramidale
+++
+++
Stratum lucidum
+
++
Subiculum
++
++
Entorhinal cortex
++
++
Amygdala
Central amygdaloid nucleus
+++
++
Medial amygdaloid nucleus
++
++
Lateral amygdaloid nucleus
++
++/+++
Basolateral amygdaloid
+++
++
nucleus
Basomedial amygdaloid
++
++
nucleus
Cortical amygdaloid nuclei
++
++
Amygdalohippocampal
++
++
area
Amygdalopiriform
++
++
transition area
Intercalated nuclei
+++
+++
Septal and basal magnocellular nuclei
Bed nucleus stria terminalis
++
++
Nucleus accumbens core
++
++
Nucleus accumbens shell
++
++
Substantia innominata
+
+
Septum lateral nucleus
Septum medial nucleus
Diagonal band of Broca
Striatopallidal system
Caudate putamen
Globus pallidus
Ventral pallidum
Claustrum
Islands of Calleja
Diencephalon
Thalamus
Anterior nuclei
Lateral nuclei
Ventral nuclei
Mediodorsal nucleus
Central nuclei
Paracentral nucleus
Parafascicular thalamic
nuclei
Paraventricular thalamic
nuclei
Geniculate nuclei
Subgeniculate nucleus
Reticular thalamic nucleus
Posterior nuclear group
Paratenial nucleus
Rhomboid nucleus
Ethmoid nucleus
Reuniens
Subthalamic nucleus
Zona incerta
Epithalamus
Habenula medial
Habenula lateral
Hypothalamus
Median eminence
Arcuate nucleus
Supraoptic nucleus
Paraventricular nucleus
Periventricular nucleus
Suprachiasmatic nucleus
Mammillary nuclei
Fornix
Mesencephalon
Midbrain
Superior colliculus
Inferior colliculus
Interpeduncular nuclei
Substantia nigra
Pars reticulata
Pars compacta
Periaqueductal gray
Rhombencephalon
Cerebellum
Molecular layer
++
++
++
++
+++
++/++
+/++
+++
+++
+++
+/++
+++
+-
+++-
-
-
++
+
+/++
++
+-
++
++
+
-/++
+
+
+
+
+/++
+
+++-/+
+++
-
+
+-/+
++++-/+
-
+
++-/+
+
++
+
+
+
+
+
+
+
+
53
Chapter 2
Table 1 - Continuation
Brain region
Cterm
Rhombencephalon
Cerebellum
Purkinje cell layer
+
Granule cell layer
+Deep cerebellar nuclei
+Pons and medulla oblongata
Locus coeruleus
+
Reticular nuclei
+Ambiguus nucleus
+Nucleus of the solitary tract
+Inferior olive
+
Scoring was classified as absent (-), low (+-),
moderate (+), high (++), very high (+++).
Nterm
+
+++
+++
Table 2 - Mean optical density of AKAP150 protein
in various compartments of the mouse central
nervous system.
Brain region
O.D.
±SEM
Telencephalon
Olfactory system
Olfactory tubercle
0,52
0.006
Cerebral cortex
Layer I
0.52
0.020
Layer II
0.46
0.026
Layer III
0.41
0.027
Layer IV
0.41
0.022
Layer V
0.23
0.018
Layer VI
0.30
0.014
Barrel cortex
0.52
0.023
Hippocampal formation
Dentate gyrus
Granule cell layer
0.16
0.019
Molecular layer
0.42
0.046
CA1 region
Stratum radiatum
0.37
0.054
Stratum oriens
0.37
0.003
Stratum lacunosum
0.21
0.035
moleculare
Stratum pyramidale
0.18
0.020
CA3 region
Stratum oriens
0.39
Stratum pyramidale
0.32
Stratum lucidum
0.10
Amygdala
Central amygdala
0.33
Basolateral amygdala
0.29
Lateral amygdala
0.33
Septal and basal magnocellular nuclei
Septum lateral nucleus
0.32
Septum medial nucleus
0.14
Striatopallidal system
Caudate putamen
0.46
Diencephalon
Thalamus
Lateral nuclei
Hypothalamus
Dorsomedial hypothalamus
0.018
0.024
0.015
0.011
0.004
0.015
0.012
0.009
0.015
0.16
0.003
0.09
0.011
Rhombencephalon
Cerebellum
Molecular layer
0.17
0.002
Granule cell layer
0.13
0.005
Values represent mean of optical density and ±
S.E.M. of each brain compartment (n=5). O.D.=
optical density.
Discussion
General overview of AKAP150 distribution in the mouse brain
Our results showed that AKAP150 is widely distributed throughout the mouse brain.
However, clear differences between brain compartments were observed. Both Western
54
AKAP150 in the mouse brain
blotting and immunohistochemistry showed the highest expression levels of AKAP150 in
striatum and cerebral cortex. In addition, relatively high AKAP150 expression was found in
hippocampus and olfactory bulb and low/no expression in cerebellum, hypothalamus,
thalamus and brain stem. Although the overall expression of AKAP150 in thalamus,
hypothalamus, midbrain and hindbrain was limited, a few nuclei in these regions showed a
moderate to high AKAP150 IR (e.g. reticular thalamic nucleus, ethmoid nucleus,
mammillary nuclei). Although it remains difficult to speculate on the specific function of a
brain region based solely on AKAP150 expression, a higher expression in these specific
nuclei may suggest a more prominent role of AKAP150 in these nuclei. E.g. the thalamic
reticular nucleus is a sheet of GABAergic neurons. It was recently shown that AKAP150
facilitates the phosphorylation of GABA(A) receptors by PKA which may have profound
local effects on neuronal excitation (Brandon et al., 2003).
It is interesting to note that in cholinergic areas such as the nucleus basalis, the medial
septum and the diagonal band of Broca, which are known to be involved in the modulation
of learning and memory, rather low levels of AKAP150 expression were observed.
AKAP150 expression was absent in the large brain fiber systems (e.g. fornix and corpus
callosum). With a few exceptions, our mouse AKAP150 data showed a distribution pattern
similar to that previously described for the rat brain (Glantz et al., 1992). Like in the mouse
brain, in the rat brain AKAP150 is abundantly expressed in olfactory bulb, cerebral cortex,
hippocampus and cerebellar Purkinje neurons whereas in thalamus, hypothalamus,
midbrain and hindbrain, AKAP150 is quantitatively characterized as modest or extremely
low (Glantz et al., 1992 and Lilly et al., 2005).
AKAP150 IR in the cortex, hippocampus and amygdala
In the mouse brain AKAP150 showed high levels of IR in the cortex. Dense staining was
observed throughout all cortical layers, except for moderate staining in immunoreactive
layer V. The same pattern of expression was observed in the rat neocortex (Glantz et al.,
1992). Interestingly, in the mouse barrel cortex (cortical layer IV), AKAP150 showed a
strong and characteristic staining which was not reported for the rat brain. The barrel cortex
and its afferent pathway from the facial vibrissae in rodents is often used as a model for
studying neuronal plasticity (Fox et al., 1996). Although the function of AKAP150 in the
55
Chapter 2
barrel cortex is not known, we can speculate on its involvement in mechanisms of neuronal
plasticity in this brain region.
AKAP150 was expressed in all mouse hippocampal subfields but mainly in apical and basal
dendrites of pyramidal neurons and dendrites of granule cells in the dentate gyrus. In
general these findings correspond to the rat hippocampal AKAP150 distribution as reported
previously (Glantz et al., 1992; Lilly et al., 2005). However, we also observed several
differences between mouse and rat AKAP150 expression in this region. Overall, CA1 and
dentate gyrus showed stronger IR than CA3 in the rat hippocampus (Lilly et al., 2005),
whereas the mouse hippocampus has a more homogeneous staining. On a more cellular
level the pyramidal neurons of the mouse hippocampus showed a somewhat different
staining in comparison to the rat AKAP150 IR. Mouse pyramidal neurons in the CA3
showed very high AKAP150 IR in their cell bodies, whereas cell bodies in the CA1
exhibited a much weaker staining. This specific staining for pyramidal neurons was not
reported previously for the rat brain (Glantz et. al., 1992; Lilly et al., 2005). Interestingly,
detailed observation of the mouse hippocampus indicated that interneurons might also
contain AKAP150. For example, AKAP150-positive cells that resemble interneurons
(based on shape, size and localization) were seen in the CA1-3 hippocampal area and in the
dentate gyrus, which were not previously reported in the rat hippocampus (Glantz et al.,
1992; Lilly et al., 2005). However, in the hippocampus from post-mortem human brains of
healthy individuals and lobectomy samples from patients with intractable epilepsy,
AKAP79 positive cells resembling interneurons were also reported in the CA1 area and in
the hilus (Sik et al., 2000). Double staining experiments for AKAP79 and calretin and
parvalbumin as interneuron markers demonstrated numerous double stained dendrites and
somata at the electron microscopic level of the human hippocampus (Sik et al., 2000).
Together these findings and our data suggest that AKAP79/150 may have a specific
function in these interneurons. Nevertheless, the nature of the non-principal cells, which
showed high expression levels of AKAP150 in the mouse brain, was not identified. Since
we did not use any cellular markers for interneurons, the staining could also reflect e.g.
excitatory mossy neurons.
Substantial levels of AKAP150 were demonstrated both in mouse and rat amygdala (Glantz
et al., 1992). The highest expression levels were found in central and basolateral amygdala
56
AKAP150 in the mouse brain
where dense staining marked both perikarya and fibers. This pattern of expression was also
reported previously for AKAP79 in human foetal amygdala where it was enriched in
dendrites and various neuronal cell types (Ulfig & Setzer, 1999).
AKAP150 expression parallels the expression of PKA-RIIβ and AMPA receptor
subunits
The AKAP79/150 family has a high affinity for the RIIβ-PKA subunit (Bregman et. al.,
1989). RIIβ is the predominant isoform and principal mediator of cAMP action in
mammalian central nervous system (Sarkar et. al., 1984). Ventra and colleagues showed
that in the rat neocortex and corpus striatum RIIβ mRNA levels paralleled the presence of
AKAP150 protein. Conversely, in brain areas showing low RIIβ levels such as cerebellum,
hypothalamus and cerebellum, the anchoring protein was absent (Ventra et al., 1996). In the
adult mouse brain, high RIIβ mRNA levels were shown in the neocortex, caudate-putamen,
hippocampus and reticular thalamic nuclei and a reduced level in the thalamus, midbrain
and hindbrain (Cadd & McKnight, 1989). This RIIβ mRNA pattern in the mouse brain
corresponds to our AKAP150 expression pattern confirming a parallel presence of both
proteins in specific mouse brain regions.
AKAP79/150 has been shown to regulate hippocampal AMPA receptor phosphorylation
and function (Colledge et al., 2000; Tavalin et al., 2002). Interestingly, in the murine
hippocampus high expression levels of both GluR1 and GluR2 AMPA receptor subunits
were reported in CA1-3 stratum oriens, radiatum and lacunosum moleculare and in the
stratum moleculare of the dentate gyrus (Yoneyama et al., 2004). This also parallels the
AKAP150 expression pattern we observed in this brain structure.
Concluding remarks
Overall, our results showed that AKAP150 protein is strongly expressed in numerous
mouse brain compartments, and particularly in those areas that were reported to be involved
in learning and memory processes. This supports the general notion that AKAP150 might
be involved in learning and memory processes. AKAP79/150 bound PKA was found to
play an important role in the regulation of AMPA receptor surface expression and synaptic
plasticity (Rosenmund et al., 1994). Changes in synaptic plasticity are suggested to be the
57
Chapter 2
possible mechanism underlying learning and memory processes, therefore a role of
AKAP79/150 in learning and memory can be expected. Initial evidence for a role of
AKAP150 in learning and memory came from a study by Moita and colleagues, who
reported that blocking PKA binding on AKAPs in the rat lateral amygdala leads to an
impairment of memory consolidation of auditory fear conditioning (Moita et al., 2002). In
addition, we could recently show that AKAP150 is upregulated in the mouse hippocampus
after exposing mice to a novel context and after associative learning (Nijholt et al., 2007).
In summary, we presented a detailed description of AKAP150 in discrete brain regions of
the mouse. Although we observed minor differences between AKAP150 staining in the
mouse and rat, our data suggested a characteristic pattern of AKAP150 distribution in these
two rodent species.
Acknowledgments
We thank Jan Keyser for valuable microscopy technical assistance. Part of this work was
supported
by
The
Netherlands
Organization
for
Scientific
Research
(NWO-
Vernieuwingsimpuls E.A.V.d.Z (Grant 016.021.017)).
References
Angelo, R.G., & Rubin, C.S. (2000). Characterization of structural features that mediate the tethering
of Caenorhabditis elegans protein kinase A to a novel A kinase anchor protein. Insights into the
anchoring of PKAI isoforms. Journal of Biological Chemistry , 275, 4351-62.
Brandon, N.J., Jovanovic, J.N., Colledge, M., Kittler, J.T., Brandon, J.M., Scott, J.D., & Moss, S.J.
(2003). A-kinase anchoring protein 79/150 facilitates the phosphorylation of GABA(A) receptors by
cAMP-dependent protein kinase via selective interaction with receptor beta subunits. Molecular and
Cellular Neuroscience 22, 87-97.
Bregman, D.B., Bhattacharyya, N., & Rubin, C.S, (1989). High affinity binding protein for the
regulatory subunit of cAMP-dependent protein kinase II-B. Journal of Biological Chemistry 264,
4648-4656.
58
AKAP150 in the mouse brain
Bregman, D.B., Hirsch, A.H., & Rubin, C.S. (1991). Molecular characterization of bovine brain P75,
a high affinity binding protein for the regulatory subunit of cAMP-dependent protein kinase II beta.
Journal of Biological Chemistry 266, 7207-7213.
Cadd, G., & McKnight, G.S. (1989). Distinct patterns of cAMP-dependent protein kinase gene
expression in mouse brain. Neuron. 3, 71-9.
Carr, D.W., Stofko-Hahn, R.E., Fraser, I.D.C., Cone, R.D., & Scott, J.D. (1992). Localization of the
cAMP-dependent Protein Kinase to the Postsynaptic Densities by A-Kinase Anchoring Proteins.
Journal of Biological Chemistry 24, 16816-16823.
Colledge, M., Dean, R.A., Scott, G.K., Langeberg, L.K., Huganir, R.L., & Scott, J.D. (2000).
Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex. Neuron 27, 107-119.
Dell'Acqua, M.L., Dodge, K.L., Tavalin, S.J., & Scott, J.D. (2002). Mapping the protein phosphatase2B anchoring site on AKAP79. Binding and inhibition of phosphatase activity are mediated by
residues 315-360. Journal of Biological Chemistry 277, 48796-802.
Dell'Acqua, M.L., Smith, K.E., Gorski, J.A., Horne, E.A., Gibson, E.S., & Gomez, L.L. (2006).
Regulation of neuronal PKA signaling through AKAP targeting dynamics. European journal of cell
biology 85, 627-33.
Faux, M.C., Rollins, E.N., Edwards, A.S., Langeberg, L.K., Newton, A.C., & Scott, J.D. (1999).
Mechanism of A-kinase-anchoring protein 79 (AKAP79) and protein kinase C interaction .
Biochemical Journal 343, 443-52.
Fox, K., Glazewski, S., Chen, C.M., Silva, A., & Li, X., (1996). Mechanisms underlying experiencedependent potentiation and depression of vibrissae responses in barrel cortex. Journal of PhysiologyParis 90, 263-269.
Franklin, K.B.J., & Paxinos, G., (1997). The mouse brain in stereotaxic coordinates. Academic Press,
San Diego.
Genin, A., French P., Doyere, V., Davis, S., Errington, M.L., Maroun, M., Stean, T., Truchet, B.,
Webber, M., Wills, T., Richter-Levin, G., Sanger, G., Hunt, S.P., Mallet, J., Laroche, S., Bliss, T.V.,
& O'Connor, V., (2003). LTP but not seizure is associated with up-regulation of AKAP-150.
European Journal of Neuroscience 17, 331-40.
59
Chapter 2
Glantz, S.B., Amat, J.A., & Rubin, C.S., (1992). cAMP signaling in neurons: patterns of neuronal
expression and intracellular localization for a novel protein, AKAP150, that anchors the regulatory
subunit of cAMP-dependent protein kinase II beta. Molecular Biology of the Cell 3, 1215-1228.
Glantz, S.B., Li, Y., & Rubin, C.S., (1993). Characterization of distinct tethering and intracellular
targeting domains in AKAP75, a protein that links cAMP-dependent protein kinase II beta to the
cytoskeleton. Journal of Biological Chemistry 268, 12796-804.
Hoshi, N., Langeberg, L., & Scott, J., (2005). Distinct enzyme combinations in AKAP signaling
complexes permit functional diversity. Nature Cell Biology 7, 1066–1073.
Jackson, S.M., & Berg, C.A., (2002). An A-kinase anchoring protein is required for protein kinase A
regulatory subunit localization and morphology of actin structures during oogenesis in Drosophila.
Development 129, 4423-4433.
Kennedy, M.B., (1997). The postsynaptic density at glutamatergic synapses. Trends in Neurosciences
20, 264-268.
Lilly, S.M., Alvarez, F.J., & Tietz, E.I., (2005). Synaptic and subcellular localization of A-kinase
anchoring protein 150 in rat hippocampal CA1 pyramidal cells: co-localization with excitatory
synaptic markers. Neuroscience 1, 155-163.
Lohmann, S. M., De Camilli, P., Einig, I., & Walter, U., (1984). High-affinity binding of the
regulatory subunit (RII) of cAMP dependent protein kinase to microtubule-associated and other
cellular proteins. Proceedings of the National Academy of Sciences 81, 6723-6727.
Malenka, R.C., & Bear, M.F., (2004). LTP and LD: an embarrassment of riches. Neuron 44, 5-21.
Moita, M.A., Lamprecht, R., Nader, K., & LeDoux, J.E., (2002). A-kinase anchoring proteins in
amygdala are involved in auditory fear memory. Nature Neuroscience 5, 837-838.
Nijholt, I.M., Ostroveanu, A., De Bruyn, M., Luiten, P.G.M., Eisel, U.L.M., & Van der Zee, E.A.,
(2007). Both exposure to a novel context and associative learning induce an upregulation of
AKAP150 protein in mouse hippocampus. Neurobiology of Learning and Memory, 87, 693-696.
Rosenmund, C., Carr, D.W., Bergeson, S.E., Nilaver, G., Scott, J.D., & Westbrook, G.L., (1994).
Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal
neurons. Nature 368, 853-856.
60
AKAP150 in the mouse brain
Sarkar, D., Erlichman, J., & Rubin, C.S., (1984). Identification of a calmodulin binding protein that
Co-purifies with the regulatory subunit of brain protein kinase II. Journal of Biological Chemistry
259, 9840-9846.
Scott, J.D., (1991). Cyclic nucleotide-dependent protein kinases. Pharmacology & Therapeutics 50,
123-145.
Sik, A., Gulacsi, A., Lai, Y., Doyle, W.K., Pacia, S., Mody, I., & Freund, T.F., (2000). Localization
of the A kinase anchoring protein AKAP79 in the human hippocampus. European Journal of
Neuroscience 12, 1155-64.
Skalhegg, B.S., & Tasken, K., (2000). Specificity in the cAMP/PKA signaling pathway. Differential
expression, regulation, and subcellular localization of subunits of PKA. Frontiers in Bioscience 5,
678-93.
Snyder, E.M., Colledge M., Crozier R.A., Chen, W.S., Scott, J.D., & Bear, M.F., (2005). Role for A
kinase-anchoring proteins (AKAPs) in glutamate receptor trafficking and long term synaptic
depression. Journal of Biological Chemistry 280, 16962-16968.
Tavalin, S.J., Colledge, M., Hell, J.W., Langeberg, L.K., Huganir, R.L., & Scott, J.D., (2002).
Regulation of GluR1 by the A-kinase anchoring protein 79 (AKAP79) signaling complex shares
properties with long-term depression. Journal of Neuroscience 22, 3044-3051.
Ulfig, N., & Setzer, M., (1999). Expression of a kinase anchoring protein 79 in the human fetal
amygdala. Microscopy Research and Technique 46, 48-52.
Ventra, C., Porcellini, A., Feliciello, A., Gallo, A., Paolillo, M., Mele, E., Avvedimento, V.E., &
Schettini, G., (1996). The differential response of PKA to cyclic AMP in discrete brain areas
correlates with the abundance of regulatory subunit II. Journal of Neurochemistry 66, 1752-1761.
Wong, W., & Scott, J.D., (2004). AKAP signaling complexes: focal points in space and time. Nature
Reviews Molecular Cell Biology 12, 959-970.
Yamauchi, T., (2002). Molecular constituents and phosphorylation-dependent regulation of the
postsynaptic density. Mass Spectrometry Reviews 21, 266-286.
Yoneyama, M., Kitayama, T., Taniura, H. & Yoneda, Y., (2004). Immunohistochemical detection by
immersion fixation with Carnoy solution of particular non-N-methyl-D-aspartate receptor subunits in
61
Chapter 2
murine hippocampus. Neurochemistry International 44, 413-422.
Ziff, E.B., (1997). Enlightening the postsynaptic density. Neuron 19, 1163-1174.
62