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The Journal of Neuroscience, April 1, 2003, 23(7):2634
Derangements of Hippocampal Calcium/Calmodulin-Dependent Protein
Kinase II in a Mouse Model for Angelman Mental Retardation Syndrome
Edwin J.
Weeber1,
Yong-Hui
Jiang2,
Ype
Elgersma3, 4,
Andrew W.
Varga1,
Yarimar
Carrasquillo1,
Sarah E.
Brown1,
Jill M.
Christian1,
Banefsheh
Mirnikjoo1,
Alcino
Silva3,
Arthur L.
Beaudet2, and
J. David
Sweatt1
1 Division of Neuroscience and 2 Department
of Molecular and Human Genetics, Baylor College of Medicine, Houston,
Texas 77030, 3 Department of Neurobiology, University of
California, Los Angeles, Medical Center, Los Angeles, California
90095-1763, and 4 Department of Neuroscience, Erasmus
University Rotterdam, 3000 DR Rotterdam, The Netherlands
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ABSTRACT |
Angelman syndrome (AS) is a disorder of human cognition
characterized by severe mental retardation and epilepsy. Recently, a
mouse model for AS (Ube3a maternal null mutation) was
developed that displays deficits in both context-dependent learning and hippocampal long-term potentiation (LTP). In the present
studies, we examined the molecular basis for these LTP and learning
deficits. Mutant animals exhibited a significant increase in
hippocampal phospho-calcium/calmodulin-dependent protein kinase II
(CaMKII), specifically at sites Thr286 and
Thr305, with no corresponding change in the levels
of total CaMKII. In addition, mutants show a reduction in CaMKII
activity, autophosphorylation capability, and total CaMKII associated
with postsynaptic density. These findings are the first to implicate
misregulation of CaMKII as a molecular cause for the neurobehavioral
deficits in a human learning disorder.
Key words:
Angelman syndrome; calcium/calmodulin-dependent
protein kinase II; long-term potentiation; postsynaptic density; protein phosphatase; autophosphorylation
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Introduction |
Angelman syndrome (AS) is a
neurological disorder characterized by severe mental retardation,
propensity for seizure (associated with a characteristic
electroencephalogram), and an ataxic gait (Williams et al., 1995
; Buoni
et al., 1999; Laan et al., 1999). AS is estimated to occur in 1 of
every 15,000-20,000 births and is most often caused by maternal
chromosomal deletion of 15q11-q13; however, maternal uniparental disomy
and imprinting center mutations also can cause AS (Clayton-Smith,
1993). In 1997, the Ube3a gene within the 15q11-13
chromosomal region was identified as the genetic locus for AS (Kishino
et al., 1997; Matsuura et al., 1997; Sutcliffe et al., 1997).
Ube3a codes for an E6-AP ubiquitin ligase, an enzyme involved in protein degradation through the ubiquitin-associated proteosome-mediated pathway. The E6-AP ubiquitin ligase is known to be
involved in the degradation of four proteins, including the p53 tumor
suppressor protein (Huibregtse et al., 1991), a human homolog to
the yeast DNA repair protein Rad23 (Kumar et al., 1999), the multicopy
maintenance protein 7 subunit involved in the initiation of DNA
replication (Kuhne and Banks, 1998), and E6-AP, which is a target for
itself (Nuber et al., 1998). Unfortunately, identification of these
target proteins has yet to shed light on any possible mechanism
underlying the etiology and learning and memory deficits of AS.
Since the identification of the AS locus, some progress has been made
in understanding the genetic and biochemical mechanisms underlying this
disorder. One important step forward in this endeavor was the
development of a mouse model for AS (Jiang et al., 1998). This mouse
model, generated by a maternal null mutation in the Ube3a
gene, revealed the characteristic subregion-specific paternal silencing
of the Ube3a gene in the brain. Because of imprinting, the
Ube3a gene product in the hippocampus and Purkinje cell
layer of the cerebellum is derived primarily from the maternal
copy of the gene. Importantly, the mouse model recapitulates the
brain-specific imprinting of Ube3a in humans, and also the
seizure, ataxic gait, and hippocampus-dependent abnormalities observed
in human AS.
In undertaking these studies, we reasoned that understanding the
deficits in hippocampal long-term potentiation (LTP) that we had
observed previously in the AS mouse model should facilitate identifying
the putative molecular mechanisms underlying the behavioral learning
deficits in AS. Thus, in the present studies, we examined the
hippocampal LTP deficits observed in the Ube3a
maternal-deficient (m
/p+) mice
and investigated the biochemical basis for these deficits. Ube3a maternal-deficient mice exhibited deficits in both
NMDA receptor (NMDAR)-dependent and -independent LTP, suggesting
that the source of the observed LTP deficits resides
downstream of calcium influx. These results led us to examine
calcium-dependent signal transduction pathways activated during LTP
induction. We found aberrant autophosphorylation and diminished
activity of hippocampal calcium/calmodulin-dependent protein (CaM)
kinase II (CaMKII), without a change in total CaMKII concentrations. The change in the phosphorylation state of CaMKII was coupled to a
significant reduction in CaMKII associated with postsynaptic density
(PSD). In addition, we found that activity for the major phosphatases
for CaMKII, protein phosphatase 1 (PP1) and PP2A, is significantly
reduced in
m/p+ mice.
Together, these results suggest that changes in CaMKII underlie the
hippocampal LTP deficits. More importantly, these data indicate that
alterations in calcium-dependent hippocampal CaMKII activation may play
a role in the learning and memory deficits in the
m/p+ mutant
mice and in humans with AS as well.
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Materials and Methods |
Hippocampal slice preparation and electrophysiology.
Mutants with the Ube3a null mutation were developed as
described previously (Jiang et al., 1998). Hippocampal slice
preparation and electrophysiology were performed as described
previously (Roberson and Sweatt, 1996). Briefly, hippocampal slices
(400 µM) were maintained on an interface chamber and bathed in oxygenated artificial CSF (in
mM: 125 NaCl, 2.5 KCl, 1.24 NaH2PO4, 25 NaHCO3, 10 D-glucose, 2 CaCl2, and 1 MgCl2) (1 ml/min) in an interface chamber maintained at either 25 or 32°C.
Extracellular field recordings were made in area CA1 stratum radiatum
by stimulation of the Schaffer collateral synapse. Stable baseline
synaptic transmission was established for 20 min at an intensity of
40-50% of the maximum population EPSP (pEPSP) before LTP-inducing
high-frequency stimulation (HFS). HFS consisted of one or three sets,
each set consisting of two trains of 100 Hz stimulation for 1 sec,
separated by 20 sec (NMDA-dependent LTP induction) or three trains of
200 Hz stimulation for 0.5 sec, separated by 2 min (NMDA-independent
LTP induction). Stimulus intensity of the HFS was matched to the
intensity used in the baseline recordings. Responses were monitored for
20 min before high-frequency stimulation was given to ensure a stable
baseline. Measurements are shown as the average slope of the pEPSP from six individual traces and are standardized to baseline recordings.
Tissue preparation. Mouse brains were immediately removed
and perfused in ice-cold saline (in mM: 125 NaCl,
2.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 25 D-glucose, 2 CaCl2, and 1 MgCl2,
saturated with 95% O2-5%
CO2, pH 7.4). Hippocampi were dissected and then homogenized in 2-3 ml of buffer (20 mM Tris-HCl,
pH 7.5, 1 mM EGTA, 1 mM
EDTA, 25 µg/ml aprotinin, 25 µg/ml leupeptin, 1 mM Na4P2O7,
500 µM phenylmethylsulfonyl fluoride, 4 mM para-nitrophenylphosphate, and 1 mM sodium orthovanadate). Protein concentrations
were determined using the BCA protein determination assay (BCA protein
assay reagent; Pierce, Rockford, IL).
Western blot analysis. Hippocampal tissue homogenates were
resolved on 10% SDS-polyacrylamide gels and transferred to Immobilon-P membranes. The membranes were blocked in 5% dry-milk solution for 1 hr. The primary antibody was diluted 1:3000 in the same 5% dry-milk
solution. The membranes were incubated in the primary antibody for 1 hr
at room temperature and then washed in Tween-TBS (TTBS) buffer
(50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20). The secondary
antibody was diluted 1:30,000 in 5% dry-milk solution, and the
membranes were incubated in the secondary antibody for 1 hr at room
temperature. The membranes were again washed in TTBS before being
developed using the enhanced chemiluminescence method (Amersham
Pharmacia Biotech, Arlington Heights, IL). The bands of each
Western blot were quantified with densitometry using a StudioScan
desktop scanner and NIH Image software to determine the amount of each
of the tested proteins.
Kinetic assays. CaMKII enzyme activity was measured by
quantifying incorporation of
[32P]PO4 into a
synthetic peptide substrate as described by Roberson et al. (1996).
Briefly, in vitro assays consisted of 5-10 µg of hippocampal homogenate protein resuspended in reaction buffer (in
mM: 200 Tris, 5 EGTA, 10 EDTA, and 20 Na2PO4), 100 µM autocamtide peptide substrate,
[
-32P]ATP, 0.5 mM ATP, and H2O in a final
assay volume of 50 µl. The reaction samples were incubated at 37°C
for 20 min and terminated by addition of 25 µl of stop solution (in
mM: 225 H3PO4 and 1 ATP). Two
aliquots from each sample were transferred onto Whatman P-51 paper. After drying at room temperature, the filter papers were
washed three times for 10 min in 0.25 M
H3PO4 and once for 2 min in
95% (v/v) ethanol with gentle agitation. Chromatography papers were
air dried before quantitation by liquid scintillation counting.
Autophosphorylation. CaMKII autophosphorylation assays were
performed from whole hippocampal homogenates. Homogenates were thawed
before use, and 15 µg of protein was used for each assay. Each 50 µl assay reaction was incubated on ice for 15 min with the addition
of 10 µM ATP to allow the CaMKII enzyme to
reach a state of autophosphorylation equilibrium. Reactions were
initiated with the addition of 0.5 µCi of
[-32P]ATP and 0.5 mM ATP with or without the 10 nM calmodulin and 1 mM
CaCl2. Reactions were incubated at 32°C for 3 min and stopped with the addition of SDS-containing sample buffer.
Samples were heated at 90°C for 1 min, and 25 µl was loaded onto a
7.5% SDS-polyacrylamide gel. After electrophoresis, gels were dried
and placed on Biomax film overnight at 
70°C in the presence of an
intensifying screen. Band intensities were analyzed and quantified by
densitometry using a Studioscan desktop scanner and NIH Image software.
Thr305 serum specificity. The
synthetic peptides CRRKLKGAIL-pT-TMLATRN containing the
phosphorylated threonine (corresponding to
CaMKII-Thr305) were coupled to keyhole
limpet hemocyanin and injected into rabbits (Elgersma et al., 2002).
The antisera were screened for recognition to activated CaMKII using
purified CaMKII (Sigma, St. Louis, MO) subjected to a
kinase reaction in the presence or absence of
Ca2+ and calmodulin (see Fig.
4A). No immunoreactivity was seen when blots were
probed with P-Thr305 CaMKII serum
to purified CaMKII samples incubated with PP1
(Sigma) after the autophosphorylation reaction (data not
shown). Specificity to the P-Thr305 CaMKII
site was tested by preincubation of the serum with either the synthetic
peptide phosphorylated at Thr305 or a
synthetic peptide lacking the Thr305
phosphate. Precleared serum was used to probe blots from hippocampal homogenates that were subjected to a kinase phosphorylation reaction in
the presence or absence of Ca2+ and
calmodulin (see Fig. 4A). Because of the apparent
high specificity of the serum exclusively to the
P-Thr305 CaMKII site, the serum was not
affinity purified and was directly used to probe blots of hippocampal
homogenates from wild-type (WT) and
m/p+ animals.
Postsynaptic density isolation. PSD was isolated as
described previously (Carlin et al., 1980). After decapitation, brains were removed and hippocampi were dissected and frozen on dry ice within
2 min. The speed of hippocampus isolation and flash freezing were
optimized to reduce ischemia-induced CaMKII translocation to the PSD,
as described previously by Suzuki et al. (1994). Hippocampi were
homogenized by hand using a homogenization buffer without detergents
(0.32 M sucrose, 1 mM
NaHCO3, 1 mM
MgCl2, and 0.5 mM CaCl2). The homogenates are then centrifuged at
1400 × g for 10 min. The resulting pellet is washed in
the homogenization buffer and centrifuged at 710 × g for 10 min. The supernatant from this spin is
combined with the one from the first spin, and this sample is
centrifuged at 13,800 × g for 10 min. The pellet from
this spin is then resuspended in a second homogenization buffer (0.32 M sucrose and 1 mM
NaHCO3). This sample is then placed on top of a
trilevel sucrose gradient (0.85, 1.0, and 1.4 M
sucrose). The sample is centrifuged through the gradient at 82,500 × g for 1 hr. The protein fraction between the 1.0 and 1.4 M sucrose layers is removed. A Triton solution
(1% v/v Triton X-100 in 0.32 M sucrose and 12 mM Tris-HCl, pH 8.1) and the second
homogenization buffer are added to this fraction in equal amounts. This
suspension is stirred gently and then centrifuged at 30,800 × g for 20 min. The resulting pellet is resuspended in the
second homogenization buffer and used in subsequent Western blotting experiments.
Immunohistochemistry/microscopy. Mice were perfused, and
brains were harvested as described previously (Varga et al., 2000). Forty micrometer coronal hemisections were cut on a cryostat and placed in cryoprotectant until time of experiment. Sections were first
washed three times for 10 min in TBS and then blocked for 2 hr
(blocking buffer: 1% BSA, 5% NGS, and 0.3% Triton X-100 in TBS).
Sections were then incubated in primary antibody for 24 hr (antibody
buffer: 0.25% BSA, 5% NGS, and 0.3% Triton X-100 in TBS).
Phospho-Thr286 CaMKII primary antibody
(Promega, Madison, WI) was used at 1:500. Sections were
again rinsed three times for 10 min with TBS and then incubated in
fluorescent-labeled secondary antibody for 2 hr in the dark.
Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probes,
Eugene, OR) was diluted to 1:500 in TBS. Sections were again rinsed
three times for 10 min, mounted on Plus slides, and coverslipped with
Supermount aqueous mounting medium (Biogenex). Confocal
imaging was accomplished using a FluoView FV300 confocal laser scanning
microscope on a BX50WI fixed-stage upright microscope equipped with a
FV5-ZM stepper motor and FluoView software (Olympus Optical, Melville, NY). Fluorescent images were acquired via
excitation with a krypton laser (568 nm line), a 510-550 nm bandpass
emission filter set, and a 10× or 20× objective (Olympus
Optical) using the confocal aperture suggested by the
manufacturer. Kalman accumulation averaging of three or four was used.
Maximum projection images were generated with the FluoView software.
All figures were ultimately prepared using Photoshop software
(Adobe Systems, San Jose, CA).
PP1/PP2A phosphatase assay. Phosphatase activity in whole
hippocampal homogenates was measured using a serine-threonine
phosphatase assay kit with a PP1/PP2A-specific phosphopeptide
(K-R-pT-I-R-R) (Upstate Biotechnology, Lake Placid, NY).
Free phosphate was detected after a 20 min incubation at 32°C using a
colorimetric assay and compared with a freshly prepared phosphate
standard. Specific activity was determined as the amount of picomoles
of phosphate released per minute per microgram of protein. Data
represent mean ± SEM. An analysis with a value of
p < 0.05 was considered to be statistically significant.
Data collection and statistical analysis. Hippocampal area
CA1 pEPSPs were recorded using Axon 1320 Digidata data acquisition hardware operated by Axon pClamp 8.2 software. One-way ANOVA was used
for multiple group data, and the paired Student's t test was used to evaluate differences in experimental control and mutant values. GraphPad Prism data analysis software was used for graph production, curve fitting, and statistical analysis. Data represent mean ± SEM. An analysis with a value of p < 0.05 was considered to be statistically significant.
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Results |
The AS mouse model is a powerful tool that can be effectively used
to better understand the biochemical mechanisms involved in human
maternal E6-AP deficiency. However, relating the genetic and
biochemical abnormalities observed in a mouse model for AS to humans
with AS has certain limitations. For instance, we have gained
substantial insight into the brain region-specific maternal imprinting
of Ube3a from investigation of the AS mouse model. This is
consistent with our current knowledge that the human Ube3a gene undergoes brain-specific imprinting (Rougeulle et al., 1997; Vu
and Hoffman, 1997), but the exact brain regions for Ube3a
imprinting in the human can only be inferred. However, there are many
phenotypic and morphological abnormalities that are quite similar in
the AS mouse model. For example, human newborns appear to be physically well formed, but by 12 months of age show signs of cranial growth declination and decreased brain size. Although not as prominent, the
m/p+ mice
show similar changes in head size, with reduced brain weight at 18 d of age that persists into adulthood (Jiang et al., 1998). In
addition, many other phenotypic characteristics of human AS are also
seen in the AS mouse model, such as an ataxic gait, seizure, an
abnormal electroencephalogram, and learning deficiencies. We reasoned
that, by understanding the deficits in hippocampal synaptic function
and molecular and biochemical changes in the AS mouse model, we could
identify putative molecular mechanisms contributing to the cognitive
deficits in human AS.
Recovery of Ube3a mutant LTP
Numerous examples exist of differential responses of hippocampal
CA1 LTP induction with variations in stimulus intensities, duration of
stimulation, or changes in incubation temperatures (Krelstein et al.,
1990; Muller and Lynch, 1990). A standard LTP-inducing stimulation
consisting of a 1 sec, 100 Hz stimulation elicits a long-lasting
increase in synaptic potentiation when slices are maintained at room
temperature (25°C). Alternatively, maintaining slices at a higher
temperature (30-32°C) and/or increasing the number of high-frequency
trains of stimulation can produce a potentiation that is substantially
higher in magnitude and longer lasting. Manipulating variables such as
stimulus intensity, temperature, and number of stimulations is often
used to determine the efficacy or potency of an applied drug or, as in
this case, to evaluate the severity or penetrance of a mutant genotype.
Initial studies showing hippocampal CA1 LTP deficits in the
Ube3a
m/p+
mutants (Jiang et al., 1998) were performed in an interface chamber maintained at 25°C and coupled with a modest LTP-inducing stimulus (consisting of two 1 sec trains of 100 Hz stimulation separated by 20 sec). As with any LTP deficit determined with a single LTP-induction protocol, it is unknown whether the LTP impairment in the
m/p+
mutants is caused by the inability to reach an LTP induction threshold
for a specific type of LTP, or alternatively, whether it reflects a
more severe perturbation in a signal transduction cascade absolutely
required for the induction of LTP. Therefore, we varied
temperature and LTP-induction protocols to determine the nature of the
LTP deficit in the Ube3a knock-out.
To test the possibility that the LTP deficits in
m/p+ mice
were caused by an increase in the threshold of LTP induction, LTP was
induced while maintaining hippocampal slices at increased temperatures
and increasing the number of stimulating trains. In the first
experiment, we maintained the interface chamber temperature at 32°C.
As observed previously for LTP induced at 25°C, LTP induced at 32°C
was essentially eliminated in AS mice (Fig.
1A). In the next
experiment, we combined the increase in temperature (32°C) with an
increase in the number of stimulus trains delivered to the slice.
Surprisingly, when the slice was maintained at 32°C and 100 Hz
stimulation was increased from one to three sets of HFS, LTP induction
was rescued in the Ube3a mutant (Fig. 1B). These results suggest that the LTP impairment in
Ube3a-deficient mice is primarily attributable to an
alteration in the threshold of synaptic stimulation necessary for LTP
induction to occur.
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Figure 1.
The LTP deficit in
m/p+ mice is NMDA receptor
dependent. Baseline synaptic responses of wild-type
(m+/p+) ( ) and
maternal-deficient mutant
(m/p+) ( ) animals were
measured before application of LTP-inducing HFS (indicated by
arrows) to hippocampal area CA1. A,
Slices were maintained at 32°C, and LTP was induced with a single set
of HFS consisting of two trains of 100 Hz stimulation for 1 sec
separated by 20 sec. B, Slices were maintained at
32°C, and NMDA receptor-dependent LTP was induced with three sets of
HFS, with each set separated by 10 min. C, NMDA
receptor-independent LTP was induced with three sets of HFS consisting
of 200 Hz stimulation for 1 sec separated by 4 min. The black
bar represents the application of the NMDA receptor antagonist
AP-5. All results are graphed as the percentage of potentiation
standardized to the baseline recording. Dashed lines
represent the 100% mark of baseline synaptic responses. Data represent
mean ± SEM.
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The induction of LTP is highly dependent on an influx of postsynaptic
Ca2+. The ability to rescue LTP in
m/p+ mice
with saturating HFS raises the question of whether the derangement of
LTP induction was upstream (NMDA receptor activation) or downstream of
Ca2+ influx. To help determine whether the
increase in LTP threshold observed in
m/p+
Ube3a mutants is caused by insufficient postsynaptic
Ca2+ influx, slices were stimulated in a
manner that allows calcium-dependent but NMDA receptor-independent LTP.
Thus, LTP was induced with repeated very-high-frequency stimulation
(200 Hz for 1 sec at 32°C) in the presence of the NMDA receptor
antagonist AP-5. Using this protocol, a robust, long-lasting
potentiation was induced in
m+/p+
controls, but Ube3a
m/p+
mutants exhibit a loss of potentiation by 30 min after the last stimulus (Fig. 1C). The inability of
m/p+
mutants to achieve NMDA receptor-independent LTP induction suggests that at least one locus of the observed LTP deficit resides downstream of calcium influx.
Potential protein kinase targets
The absence of E6-AP ubiquitin ligase causes the accumulation of
E6-AP targeted proteins (such as p53) in areas for which maternal gene
imprinting is occurring, but no known LTP-associated protein has been
identified as a target of E6-AP. Therefore, potential candidates for
the observed physiologic deficits downstream of calcium influx could
include any calcium-dependent, or alternatively, calcium-activated
enzyme necessary for LTP induction (Fig.
2A). Thus, in the next
phase of our experiments, we focused on four calcium-regulated protein
kinase families that have gained attention for playing prominent roles
in LTP and learning and memory in the behaving animal: protein kinase C
(PKC), cAMP-dependent protein kinase A (PKA), extracellular
signal-regulated kinase (ERK), and CaMKII. Common characteristics make
these attractive candidates for possible involvement in the
m/p+ mutant
LTP and behavioral phenotype. For example, all four kinases are (1)
activated by HFS in the hippocampus, (2) directly or indirectly affected by calcium influx into the postsynaptic neuron, (3) involved in the induction of hippocampal LTP, and (4) known to be necessary in
mammalian learning. Because the
m/p+
mutants are deficient for E6-AP ubiquitin ligase in the hippocampus, we
predicted that there would be a significant increase in the relevant
protein target of E6-AP in the mouse. Therefore, an examination was
performed of the levels of a wide variety of specific members of these
protein kinase families in the hippocampus of our
m/p+ mutant
mice.
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Figure 2.
Kinase concentrations in
m/p+ mutant hippocampus.
A, A schematic diagram of a postsynaptic neuron showing
potential candidates (shaded hexagons) for LTP and
learning disruption observed in E6-AP-deficient mice. B,
Quantitative Western blot analysis on hippocampal homogenates from
wild-type and m/p+ mutants
probed for total PKC, PKA [ catalytic (
Cat.), II regulatory (II Reg.), and  I
regulatory (I Reg.) subunits], ERK (p42 and p44 MAP
kinase), and CaMKII. C, Top,
Representative Western blot showing no change in total CaMKII but an
increase in P-Thr286 CaMKII in
m/p+ mouse hippocampal
homogenates. Bottom, Quantitative Western blot analysis
of hippocampal homogenates for protein levels of phospho-PKC,
phospho-PKA, phospho-p42 ERK, and phospho-CaMKII. A significant
increase was detected in m/p+
mice only for total P-Thr286 CaMKII (WT, 100 ± 7.1%, n = 10;
m/p+, 162 ± 16.2%,
n = 10; p = 0.0024). Results
shown are the percentage difference of
m/p+ protein levels compared
with that of wild type. Dashed line represents control level
of 100%. *p < 0.05. Data represent mean ± SEM. PLC, Phospholipase C; DAG,
diacylglycerol; MEK, mitogen and extracellular signal
regulated kinase.
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PKC/PKA/ERK/CaMKII protein levels in
m/p+ mutants
Knock-out mouse models for PKC- and PKC- make it evident
that these specific isoforms likely play important roles in mammalian synaptic plasticity and memory (Abeliovich et al., 1993a,b; Weeber et
al., 2000). Therefore, any ubiquitin ligase-dependent alterations in
PKC isoform expression could have profound effects on LTP induction and
learning. Levels of seven different PKC isoforms were unchanged in the
hippocampus of Ube3a mutants (data not shown). To test whether slight changes in each isoform could result in a significant change in the total PKC complement, a broad-spectrum antibody for total
PKC detection was used. We determined that the total PKC protein level
in m/p+
homogenates was no different from that of controls (Fig.
2B).
We were particularly interested in the levels of PKA, because
ubiquitin-proteosome-dependent reductions in the regulatory subunit
concentrations of PKA are required for long-term facilitation in
Aplysia as part of a mechanism by which autonomously active PKA is generated (Chain et al., 1999). In addition, PKA is known to be
important in triggering the late phase of LTP and in long-term memory
in mice (Chetkovich et al., 1991; Roberson and Sweatt, 1996; Abel et
al., 1997). However, we found no difference in the (catalytic) and
I or II (regulatory) subunits of PKA in the hippocampus of AS
mice (Fig. 2B). This suggests that E6-AP is not
involved in PKA regulatory or catalytic subunit degradation, and that
this site is not the locus of derangement in
m/p+ mice.
It has only recently become clear that the ERK cascade is essential for
the induction of NMDA receptor-dependent and -independent LTP and plays
a critical role in mammalian learning (Adams and Sweatt, 2002). This
prompted us to examine the protein levels of hippocampal p42 and p44
ERK levels in the
m/p+
Ube3a mutant. Hippocampal homogenates from Ube3a
mutant animals probed for p42 and p44 ERK MAP (mitogen-activated
protein) kinase immunoreactivity showed no change in protein
levels compared with wild-type controls (Fig.
2B).
CaMKII is particularly enriched in the brain and exhibits
multifunctional roles in calcium-mediated signal transduction
processes. CaMKII is known to be involved in synaptic plasticity and
learning. Neuronal CaMKII is predominately composed of the and subunits, with sizes of 52 and 60 kDa, respectively. The regulation of
CaMKII is complex, and is dependent on many factors, including calcium influx, subunit composition, binding to the postsynaptic density, calmodulin availability, and the autophosphorylation state of the
holoenzyme. These characteristics make this enzyme especially susceptible to changes within the cellular milieu. However, no changes
in total CaMKII concentrations were seen in AS mice (Fig. 2B). Thus, the results on levels of specific PKC,
PKA, ERK, and CaMKII isoforms strongly suggest that protein levels of
several kinases known to be involved in synaptic plasticity and
learning are normal in the AS hippocampus.
Hippocampal protein phosphorylation
Although an absence of significant changes in protein
concentration were observed for PKC, PKA, ERK, and CaMKII, changes in the phosphorylated form of these kinases may not be evident by total
protein determinations. The important regulatory actions of
phosphorylation are well established, especially regarding the
regulation of protein kinase activity. Such is the case in each of the
kinases we investigated. Alterations in levels of phosphorylated
protein kinases may underlie the disruption in LTP and learning in the
AS mouse. Moreover, there are several examples of protein
phosphorylation acting as a molecular switch to target a protein for
ubiquitination and degradation through the proteosome pathway (Basu and
Haldar, 2002; Zheng et al., 2002). Thus, phosphorylated kinases
involved in LTP may be a target of E6-AP. To test this hypothesis, we
examined the level of phosphorylated PKC, PKA, ERK, and CaMKII. No
changes were seen in the phosphorylation levels of PKC, PKA, or ERK
(P42) isolated from hippocampal homogenates of
m/p+ mice.
However, a significant increase was detected in phospho-CaMKII at
the autophosphorylation site threonine 286 (Thr286) (Fig. 2C).
These data suggest that alterations in CaMKII activity, secondary to
altered autophosphorylation, could contribute to the LTP and learning
phenotype of the AS mouse. This observation becomes increasingly
important as it has been widely demonstrated, using genetic and
pharmacological manipulations, that normal CaMKII function is necessary
for learning and memory. Moreover, the alteration in CaMKII
autophosphorylation appears to be a specific and restricted phenomenon.
Importantly, absence of E6-AP in the hippocampus of m/p+ mice
does not affect the overall phosphorylation state of any of the other
proteins we tested. These results highlight the specific nature of the
hippocampal derangement associated with the Ube3a maternal-deficient phenotype.
Autophosphorylation assays
To confirm the changes in CaMKII autophosphorylation, we
performed another experiment using a back-phosphorylation approach. Our
rationale for this experiment is as follows: the result showing an
increase in hippocampal P-Thr286 CaMKII
in our Ube3a maternal-deficient mice suggested that sites for additional autophosphorylation would no longer be available. Therefore, we performed an autophosphorylation assay in which CaMKII-activating concentrations of Ca2+
and CaM were added to hippocampal homogenates, and the amount of
subsequent additional autophosphorylation was quantified by assessing
32P incorporation from
[32P]ATP. Figure
3A shows that the amount of
Ca2+/CaM-induced CaMKII
autophosphorylation was significantly reduced in hippocampal
homogenates from our Ube3a-deficient mice. An important caveat to keep in mind when assessing these results is that this assay
does not give an indication of which sites are autophosphorylated [i.e., the assay does not distinguish between autophosphorylation at
threonine 286 versus threonine 305 or
Ser314 (Patton et al., 1990) of the catalytic subunit]. Nevertheless, this observation supports the
Western blot analysis results showing that increased autophosphorylated
CaMKII is present in the Ube3a maternal-deficient mouse
hippocampus.
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Figure 3.
Increased phosphorylated Thr286
CaMKII corresponds to reduced post hoc
autophosphorylation and kinase activity. A,
Top, Representative autoradiograph from hippocampal
homogenates corresponding to and catalytic subunits of CaMKII
in the presence of [-32P]ATP before and after
Ca2+/calmodulin stimulation. Bottom,
Quantitative analysis of the total
Ca2+/CaM-dependent CaMKII autophosphorylation in
hippocampal homogenates of wild-type animals ( ) or
m/p+ mutants ( ) shows a
significant reduction in total CaMKII autophosphorylation after
activation with calcium and calmodulin (wild type, 100 ± 5.5%,
n = 6;
m/p+, 71 ± 8.9, n = 7; p = 0.002).
Bars represent the percentage of quantified signal
compared with that of wild types. B, Hippocampal
homogenates from wild-type animals () or
m/p+ mutants () were assayed
for basal CaMKII activity and CaMKII activity after activation with
Ca2+ and CaM. The
m/p+ mutants
(n = 6) show no significant difference in basal
phosphotransferase activity compared with wild-type controls
(n = 7). Lower total activity after CaMKII
activation in the presence of Ca2+ and CaM was seen
in m/p+ mutants (wild type,
0.33 ± 0.05, n = 6;
m/p+, 0.487 ± 0.04, n = 7; p = 0.048). Data
represent mean ± SEM. N/D, Not detected;
Wt, wild type. *p < 0.05.
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In vitro CaMKII assay
Although the activity of CaMKII is highly sensitive to calcium
influx, the activity of the enzyme and its sensitivity to successive increases in calcium concentrations are altered after
autophosphorylation. Changes in the phosphorylation state of the subunit of CaMKII can alter the kinetic properties of the holoenzyme,
control translocation, and modify enzyme function, all without a
detectable change in total CaMKII concentrations. Our observation of an
altered phosphorylation state of CaMKII suggests a resulting change in
CaMKII enzymatic activity. Specifically, a greater ratio of
P-Thr286 CaMKII to
nonautophosphorylated CaMKII enzyme would suggest an increased basal
calcium/calmodulin-independent activity. The increase in autonomously
active CaMKII complement should also translate into a reduced
fold-increase in activity in response to activation with
Ca2+ and CaM. We tested this hypothesis by
performing in vitro CaMKII activity assays using hippocampal
homogenates and comparing the amount of basal activity and
Ca2+/CaM-stimulated activity between
wild-type and
m/p+
animals. The maternal-deficient mice showed a reduced, but not quite
significant, basal CaMKII phosphotransferase activity (Fig. 3B). Moreover, assays performed in the presence of calcium
and calmodulin revealed a significant reduction in the
phosphotransferase activity of the maternal-deficient mutants (Fig.
3B). No differences are seen when the percentage of
activation is determined (percentage change of basal vs activated
phosphotransferase activity) (data not shown). These data demonstrate
that the increases in the autophosphorylation state of CaMKII
associated with E6-AP deficiency cause an overall reduction in
CaMKII activity, most noticeably as a significant reduction in response
to Ca2+ and CaM.
Thr305-CaMKII autophosphorylation
Our in vitro CaMKII activity results were unexpected.
We hypothesized that a greater
P-Thr286-CaMKII complement would
translate into a greater overall basal CaMKII activity in the absence
of Ca2+ and CaM. One explanation for these
results is that a subset of CaMKII in the assay is in an inactive
state. Previous in vitro experiments indicate that
autophosphorylation of Thr305 and
Thr306 sites on and CaMKII,
respectively, occurs after
Thr286-CaMKII phosphorylation under
some circumstances (Colbran and Soderling, 1990; Patton et al., 1990;
Mukherji and Soderling, 1994). This can affect both the
Ca2+/CaM-independent activity (Lou and
Schulman, 1989) as well as the
Ca2+/CaM-dependent activity (Kuret and
Schulman, 1985; Hashimoto et al., 1987; Lickteig et al., 1988; Lou and
Schulman, 1989; Mukherji and Soderling, 1994).
Therefore, we hypothesized that increased phosphorylation of
Thr305 CaMKII
(P-Thr305 CaMKII) was occurring in the
AS mice as an explanation for the decreased basal enzyme activity
despite the increase in Thr286-CaMKII
autophosphorylation. We tested this hypothesis using an antiserum
raised against a peptide duplicate of the phosphorylated Thr305,
Thr306 CaMKII site for the and CaMKII subunits, respectively (CRRKLKGAIL-pT-TMLATRN). It
should be noted that, because of the high homology between the and
subunits of CaMKII, the peptide used resulted in an antiserum that
was able to recognize both the Thr305
CaMKII and Thr306 CaMKII subunits
(Fig. 4). Figure 4A
shows the specificity of the serum to the phosphorylated
Thr305/306 CaMKII site. Purified CaMKII
(Sigma) was placed in a kinase reaction mixture in the
presence or absence of Ca2+ and CaM
(described in Materials and Methods). Western blot analysis using the
Thr305/306 CaMKII serum showed
immunoreactivity specifically to the activated CaMKII. We then tested
for specificity to the P-Thr305/306 CaMKII
site using hippocampal homogenates from control wild-type mice. Again,
homogenates were placed in a kinase reaction with or without
Ca2+ and CaM, and Western blot analysis
was performed using the Thr305/306 CaMKII
serum. Both the and subunits of CaMKII were detected using the
P-Thr305/306 CaMKII serum to probe
hippocampal homogenates from wild-type animals (Fig.
4A). The wild-type control samples (no peptide
preincubation) show immunoreactivity corresponding to the and subunits of CaMKII, which is increased in the homogenate after
activation with Ca2+ and CaM.
Immunoreactivity is nearly absent if the
Thr305/306 CaMKII serum is preincubated
with a phosphorylated Thr305/306 peptide
(Fig. 4A), and no change is seen in immunoreactivity if serum was preincubated with the nonphosphorylated synthetic peptide
(data not shown). These results suggest that the
Thr305/306 CaMKII serum is highly
sensitive to the autophosphorylated species of CaMKII, is specific to
the Thr305/306 CaMKII autophosphorylation
site, and is reliable to test for changes in
P-Thr305 CaMKII in hippocampal
extracts.
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Figure 4.
Increased phosphorylated Thr305
CaMKII in m/p+ mutants.
A, Left blot, Western blot analysis
showing that P-Thr305/306 CaMKII serum recognizes
purified CaMKII activated with Ca2+/CaM, but not
nonactivated CaMKII. Right blot, Specificity for the
Thr305 autophosphorylation site was tested with
autophosphorylation reactions with hippocampal homogenates from
wild-type mice in the absence [control (Ctrl)]
or presence
(+Ca2+/CaM)
of calcium and calmodulin. Control Western blots show an increase in
immunoreactivity for activated CaMKII corresponding to the and subunits of CaMKII. Addition of antigen completely blocked recognition
of phospho-Thr305/306 CaMKII in control and
activated homogenates (peptide block). B,
Top, Representative Western blot analysis of hippocampal
homogenates probed for P-Thr305/306 CaMKII
(right blot). The same blot was then stripped and
reprobed for total CaMKII (left blot).
Bottom, Quantitative Western blot analysis shows no
change in total CaMKII but an increased Thr305
CaMKII phosphorylation in
m/p+ mutants in whole
hippocampal homogenates [wild type (Wt), 100 ± 12, n = 7;
m/p+, 133.2 ± 3.3, n = 7; p = 0.02]. Data
represent mean ± SEM. *p < 0.05.
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Using the Thr305/306 CaMKII serum, we
found that mice maternally deficient for Ube3a showed a
significant increase in Thr305 CaMKII
autophosphorylation in extracts of hippocampal homogenates (Fig.
4B). Thus, the
m/p+
mutants exhibit altered phosphorylation of CaMKII at both sites of
autophosphorylation, Thr286 (Fig.
2C) and Thr305 (Fig.
4B). The increase in
P-Thr305 CaMKII is consistent with the
observed reduction in basal CaMKII activity in our in vitro
assays and also with the loss of
Ca2+/CaM-induced CaMKII activation, and
indeed appears to be sufficient to explain the observation.
Postsynaptic density isolation
Recently, a T305D CaMKII-transgenic mouse was generated that
mimics persistent inhibitory phosphorylation. This mutation is capable
of decreasing the association of CaMKII with the PSD and can
detrimentally affect LTP and learning (Elgersma et al., 2002). In
contrast, blocking inhibitory phosphorylation increases CaMKII in the
PSD. These remarkable results raise the interesting question of whether
increased Thr305-phosphorylated CaMKII
in our m/p+
mice could result in decreased PSD-associated CaMKII. To test this
hypothesis, we examined levels of total and autophosphorylated CaMKII
from PSDs isolated from the hippocampus of wild-type and m/p+ mice.
We found that, in our
m/p+
mutants, the total PSD-associated CaMKII was significantly reduced, to
nearly one-half of that found in wild types (Fig.
5A). These results suggest
that in the AS mice, there are sufficient amounts of phosphorylated
Thr305 CaMKII to cause a considerable
translocation of the CaMKII complement away from the PSD. To test
further the phosphorylated state of the CaMKII present in the PSD, we
probed for P-Thr286 and
P-Thr305 CaMKII. Surprisingly, we found a
2.5-fold increase in P-Thr286 CaMKII but
no change in P-Thr305 CaMKII (Fig.
5B). These results support recent data showing that CaMKII autophosphorylated at Thr305/306
has lower affinity for the PSD than nonphosphorylated CaMKII (Strack
et al., 1997a; Shen et al., 2000).
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Figure 5.
PSD-associated CaMKII and autophosphorylated
CaMKII. A, B, Top,
Representative Western blot for PSD-associated total CaMKII
(A) and P-Thr286 and
P-Thr305 CaMKII (B).
A, Bottom, PSD fractions isolated from
the hippocampus of WT () or
m/p+ mutants () show a
reduction of ~50% in total PSD-associated CaMKII (WT, 100.0 ± 4.2, n = 3;
m/p+, 54.6 ± 6.7, n = 4; p = 0.045).
B, Bottom, P-Thr286
CaMKII shows an ~2.5-fold increase in the PSD fraction (WT,
100.0 ± 1.9, n = 3;
m/p+, 264.3 ± 36.4, n = 4; p = 0.012).
P-Thr305 CaMKII levels in the PSD were detectable
but showed no significant differences between wild-type and
m/p+ mice. Data represent
mean ± SEM. *p < 0.05.
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A caveat to these results arises because of the ability of CaMKII to
rapidly translocate to the PSD after animals have been killed
(Suzuki et al., 1994). Suzuki et al. (1994) have reported that, in rat
frontal cortex, the amount of CaMKII is 2.7, 4.0, 7.8, 8.6, or 9.4%
of the total PSD protein at 0, 2, 5, 30, or 60 min after decapitation.
To minimize the potential of postkilling CaMKII translocation,
we rapidly removed and flash froze hippocampi within 2 min of
decapitation. It is believed that an ischemia-induced chemical
modification renders the enzyme insoluble and causes the association of
CaMKII with the PSD; however, the exact mechanism underlying the
translocation is unknown. Thus, it is difficult to link changes in
PSD-associated CaMKII in mice deficient for hippocampal E6-AP with this
phenomenon. Despite this caveat, our observations of an increased
concentration of P-Thr286 CaMKII in
PSDs from maternal-deficient mice support findings by Elgersma et al.
(2002) of the subsequent Thr305
phosphorylation step promoting PSD dissociation. These observations also indicate that not all
Thr286-phosphorylated CaMKII enzyme is
also phosphorylated at Thr305, suggesting
that the Thr286-autophosphorylated kinase
does not automatically undergo subsequent phosphorylation at
Thr305. Overall, these results are in
remarkable agreement with the observations reported by Elgersma et al.
(2002) and are consistent with our hypothesis of altered
Thr305 autophosphorylation in the AS mouse model.
Immunohistochemistry
Immunohistochemical techniques were used to localize the changes
seen in immunoreactivity to phosphorylated CaMKII at
Thr286. Because we had seen a significant
increase in Thr286-autophosphorylated
CaMKII in our Western blot analysis of whole hippocampal homogenates
(Fig. 2C) and in an isolated PSD fraction (Fig.
5B), we used immunohistochemistry to determine whether there might be qualitative differences in the cellular distribution of
hippocampal CaMKII in AS mice. As shown in Figure
6, phosphorylated Thr286 CaMKII is localized in the
wild-type mouse under basal conditions most strongly in the pyramidal
cell layer of area CA1 as well as the granule cell layer of the dentate
gyrus. The greatest increase in immunoreactivity is seen in the
pyramidal cells in area CA3 (Fig.
6E,F). Weaker
immunoreactivity is seen in the dendritic layers of these cells,
including the stratum oriens and stratum radiatum of areas CA1 and CA3
and the molecular layer of the dentate gyrus. The distribution of
phosphorylated CaMKII at Thr286 in an
Angelman mouse hippocampus appears qualitatively the same as that of a
wild-type mouse, yet the total immunoreactivity is increased across
all labeled areas. Overall, these data suggest that the increase in
autophosphorylated CaMKII in AS mice is not specifically localized to
one cellular subregion.
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Figure 6.
Increased immunoreactivity to phosphorylated
CaMKII at Thr286 in Angelman mouse hippocampus.
Phosphorylated CaMKII at Thr286 was detected
immunohistochemically in the hippocampi of wild-type (A,
C, E) and Angelman (B,
D, F) mice. Increases in
immunoreactivity are seen in the stratum pyramidale, stratum
oriens, and stratum radiatum of CA1 and CA3 as well as the molecular
layer and granule cell layer of the dentate gyrus. A,
B, 100× magnification of the hippocampus.
C, D, 200× magnification of area CA1.
E, F, 200× magnification of area
CA3.
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Protein phosphatases PP1/PP2A
The observed increases in autophosphorylated CaMKII suggest that a
causative candidate for this disruption would be a disruption of a
CaMKII phosphatase. Previous investigation has revealed the necessity
for phosphatase activity in the regulation of neuronal plasticity and
memory formation (for review, see Winder and Sweatt, 2001). A group of
these phosphatases, such as PP1 and PP2A, exhibit high CaMKII-specific
phosphatase activity and are associated with LTP and learning (Strack
et al., 1997b; Blitzer et al., 1998; Kasahara et al., 1999; Brown et
al., 2000; Bennecib et al., 2001; Winder and Sweatt, 2001). Therefore,
we sought to determine whether alterations in the levels of the
major CaMKII phosphatases, PP1 and PP2A, were responsible for the
increase in the P-Thr286 CaMKII
species. We found no significant difference in PP1 or PP2A total
protein levels in hippocampal homogenates from
m/p+ mice
(Fig. 7A). These results
indicate that Ube3a deficiency does not affect protein
levels of two known phosphatases associated in
P-Thr286 CaMKII dephosphorylation.
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Figure 7.
Decreased PP1/PP2A activity in
m/p+ mutants. A,
Quantitative Western blot analysis showing no change in total protein
levels of PP1 and PP2A from hippocampal homogenates of
m/p+ mutant animals compared
with WT controls. The dashed line indicates wild-type
protein levels. B, Phosphatase activity was measured for
PP1/PP2A using a PP1/PP2A-specific phosphopeptide (K-R-pT-I-R-R).
Phosphatase activity was significantly reduced in the
m/p+ mutants () (0.659 ± 0.08 pmol · min1 · µg1
protein; n = 5) compared with wild-type controls
() (1.758 ± 0.27 pmol · min1 · µg1
protein; n = 8; p = 0.009).
Data represent mean ± SEM. *p < 0.05.
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In a final series of experiments, PP1/PP2A phosphatase activity was
assayed to determine whether the increase in CaMKII autophosphorylation was a result of alterations in phosphatase capacity. Phosphatase activity was measured using a PP1/PP2A-specific phosphopeptide (K-R-pT-I-R-R). Phosphatase activity using this assay system was found
to be significantly reduced in the
m/p+
mutants (0.659 ± 0.08 pmol · min1 · µg1
protein; n = 5) compared with controls
(1.758 ± 0.27 pmol · min1 · µg1
protein; n = 8; p = 0.009) (Fig.
7B). The prodigious 2.5-fold decrease in PP1/PP2A
phosphorylase activity in the
m/p+
mutants strongly suggests that the aberrant state of
P-Thr286 and
P-Thr305 CaMKII phosphorylation is
attributable to changes in the activity of one or both of these
important phosphatases. This observation also suggests that the
increase in Thr305 CaMKII is
attributable to both the increase in
P-Thr286 CaMKII, because
phosphorylation at Thr286 precedes
phosphorylation at Thr305 (Colbran and
Soderling, 1990; Hanson and Schulman, 1992), and reduced PP1 and/or
PP2A activity, which are known to dephosphorylate P-Thr305 CaMKII in vitro
(Patton et al., 1990).
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Discussion |
Consistent with the hypothesis that altered LTP parallels a
learning deficit in mice and humans, we show that a mouse model for a
human mental retardation syndrome displays pronounced hippocampal LTP
deficits. Furthermore, the finding that CaMKII is perturbed, without
changes in other learning-related kinases, suggests CaMKII as the site
for the decreased plasticity and learning and memory defects. These
results are consistent with the current literature implicating a role
for CaMKII in learning, but extend this to include human learning as
well. We reason that our mouse model reflects the mechanisms that are
in place in children afflicted with AS. This is supported by our
knowledge that the maternal imprinting of Ube3a occurs in
both systems, and that this imprinting is restricted to the brain in
both humans (Rougeulle et al., 1997; Vu and Hoffman, 1997) and mice
(Jiang et al., 1998). Finally, the CaMKII-dependent LTP deficit appears
to be caused by altered CaMKII regulation in the hippocampus,
specifically because of alterations in the autophosphorylation state of
the enzyme. These data thereby implicate CaMKII as being required for
normal learning in humans.
Insights into the LTP defects in Ube3a maternal-deficient mice
The necessity of CaMKII for synaptic plasticity is well
established. The induction of NMDA receptor-dependent LTP requires CaMKII activation in the postsynaptic neuron (Nicoll and Malenka, 1995;
Lisman et al., 1997), and mice deficient for CaMKII show deficits in
hippocampal LTP (Silva et al., 1992; Hinds et al., 1998). The sites of
CaMKII autophosphorylation are also important in LTP induction.
Mutations of the Thr286 site to prevent
autophosphorylation or, conversely, to produce a calcium-independent
form of CaMKII, result in LTP deficits for some types of LTP
inducing stimulation (Giese et al., 1998). With the recent production
of a knock-in mouse with mutations that either mimic or prevent
inhibitory phosphorylation comes the first insight into the physiologic
role of inhibitory autophosphorylation of CaMKII in synaptic plasticity
and learning (Elgersma et al., 2002). These studies reveal that T305D
mutants show a significant LTP deficit in studies using theta burst
stimulation and 100 Hz, 1 sec HFS protocols. These results are
especially exciting because we are investigating effects of both
phospho-Thr286 and
Thr305 CaMKII increases that, because of
maternal imprinting of the Ube3a gene, are nearly exclusive
to the hippocampus. We conclude that the derangements in CaMKII
autophosphorylation at Thr286 and
Thr305 contribute to the overall AS
phenotype. Indeed, the results of Elgersma et al. (2002) suggest that
the specific molecular change in
phospho-Thr305 CaMKII is sufficient to
explain the LTP and learning deficits seen in the AS mouse model.
Regulatory consequences of altered CaMKII phosphorylation
What are the potential effects associated with an increased
Thr286 and
Thr305 phosphorylation in CaMKII in
m/p+
mutants? In a population of CaMKII molecules in which
Thr286 and
Thr305 are chronically phosphorylated, one
would predict that the overall effect would be reduced basal activity,
because of Thr305 phosphorylation, and
insensitivity to increased Ca2+/CaM
concentrations, because of the combination of
Thr286 and
Thr305 autophosphorylation. This scenario
is in agreement with our in vitro assay results, which do
not show an increase in basal activity in the absence of
Ca2+/CaM, despite the presence of
increased P-Thr286 CaMKII. Also in
agreement is the reduced activity in the presence of
Ca2+/CaM, which could reflect both the
Ca2+/CaM-insensitive
Thr286 and inhibited
Thr305 CaMKII complement. Moreover, this
may also explain the hippocampal LTP deficit when using a modest
LTP-inducing HFS protocol, but in which there is normal LTP with
saturating HFS. A compromised, but not absent, CaMKII signal in
CA1 may result in an inability of LTP induction with the application of
a modest stimulus input. However, if an LTP-inducing protocol is
robust, the result is sufficient CaMKII activation needed to trigger
LTP induction.
Altered PSD-associated CaMKII
The potential regulatory problems involved with the aberrant
autophosphorylated CaMKII are compounded in light of recent studies focusing on CaMKII autophosphorylation and enzyme translocation. In a
series of elegant studies by Shen and colleagues (Shen and Meyer, 1999;
Shen et al., 2002), it was found that the activation of NMDA
receptors results in translocation of CaMKII from the cytosol to the
postsynaptic density regions (Shen and Meyer, 1999). Also, HFS triggers
a transient translocation of CaMKII from the cytosol to the PSD that is
primarily dependent on the autophosphorylation state of the CaMKII at
Thr286 (Shen et al., 2000). Results by
Elgersma et al. (2002) reveal that a T305D CaMKII mouse, which mimics
persistent inhibitory phosphorylation, shows decreased association of
CaMKII with the PSD. Conversely, a TT305/6VA CaMKII mutation results in
greater PSD-associated CaMKII. Together, these results are consistent with published reports that P-Thr286
CaMKII is localized to the PSD, whereas
P-Thr305 CaMKII causes disassociation
from the PSD. Our results suggest a derangement of this regulatory
mechanism in our AS mouse model.
Our results show that the total pool of CaMKII in AS mice has more
Thr286 and
Thr305 autophosphorylation. However, the
PSD-associated fraction of CaMKII shows increased
Thr286 phosphorylation but not increased
Thr305 phosphorylation. This supports the
model that Thr286 phosphorylation promotes
PSD association by binding to the NMDAR and is in agreement with the
observation that association with the NMDAR effectively blocks
inhibitory phosphorylation (Bayer et al., 2001) and that
Thr305 phosphorylation promotes PSD
dissociation (Elgersma et al., 2002). This situation does not preclude
there being a population of unaffected CaMKII available for normal
signaling and activation, supported by our saturating HFS protocol LTP
results. The in vitro activity results showing CaMKII
activation in the
m/p+
mutants, albeit reduced total activation, is also consistent with this
idea. Regardless, the alterations seen for both CaMKII autophosphorylated regulatory sites
(Thr286 and
Thr305) may represent a cumulative
disruption in CaMKII signaling capability in the hippocampus. This may
explain why the behavioral and LTP deficits in the E6-AP
maternal-deficient mice are more pronounced than those in the
transgenic mice described by Elgersma et al. (2002).
Protein phosphatase activity and altered
autophosphorylated CaMKII
Our data support the hypothesis that CaMKII is not a target of
E6-AP-dependent degradation but is indirectly affected by E6-AP deficiency, as observed by the significant increase in the
P-Thr286 and
P-Thr305 CaMKII without an increase in
total CaMKII complement. The considerable reduction in PP1/PP2A
phosphatase activity suggests that the changes in CaMKII
phosphorylation are a direct result of reduced protein phosphatase
activity, specifically PP1 and/or PP2A. What may be the underlying
mechanism of reduced PP1/PP2A activity? The observed alterations in
P-Thr286 and
P-Thr305 CaMKII may be caused by the
endogenous alterations associated with hippocampal E6-AP deficiency,
specifically an increase in proteins usually targeted by a now-absent
ubiquitin ligase. For example, p53 is a known target of E6-AP and
grossly accumulates in the hippocampus of
m/p+ mice,
as well as in humans with AS. An interesting possibility arises from
the identification of a p53-associated protein, designated p53 binding
protein 2 (p53BP2), which can bind p53 and PP1 in a mutually exclusive
manner (Helps et al., 1995). Very little is known about p53BP2, and its
physiological importance in the CNS is unclear. However, it is known
that p53BP2 can inhibit the phosphatase activity of PP1 at nanomolar
concentrations. It is hypothesized that p53BP2 may be involved in the
dephosphorylation and regulation of p53, but its effect on CaMKII
phosphorylation is completely unknown at present. This raises the
interesting possibility that the chronic presence and considerable
increase in p53 in the imprinted areas of AS mice and humans can
directly affect PP1 activity through p53BP2. The effect of p53BP2 may
occur either through an upregulation of p53BP2 in response to p53
levels or through a change in the localization of p53, and subsequently p53BP2, throughout the neuron. An alternative hypothesis is that phosphorylation of copious p53 protein can cause phosphatase
inhibition. This hypothesis is supported by recent research showing
that inhibition of PP1 can occur after p53 phosphorylation in rat
cardiomyocytes (Long et al., 2002). The actions of p53BP2 or p53
phosphorylation in Thr286 CaMKII
dephosphorylation represent an exciting area of future research.
Conclusions
The association of the observed neurobehavioral features of AS is
consistent with our current knowledge of the role of CaMKII in
mammalian neural processes. The most important implication of our
results relates to learning in humans. Various experimental data
support the presupposition that the AS mouse model accurately reflects
the condition in vivo in AS children. Our data support the
hypothesis that the loss of hippocampal LTP underlies the learning
deficit in humans. Finally, our observations support the hypothesis
that the Ube3a maternal deficient-associated increase in the
P-Thr286 and
Thr305 CaMKII may underlie the deficits in
synaptic plasticity and learning in our AS mouse model, and potentially
underlie the etiology of human AS. This study is significant because it
represents the first report that an alteration in CaMKII has the
potential to cause a human disorder associated with a severe learning deficiency.
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FOOTNOTES |
TOP
ABSTRACT
Introduction
