 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 15, 2001, 21(16):6000-6007
A High-Efficiency Protein Transduction System Demonstrating the
Role of PKA in Long-Lasting Long-Term Potentiation
Masayuki
Matsushita1,
Kazuhito
Tomizawa1,
Akiyoshi
Moriwaki1,
Sheng-Tian
Li1,
Hiroaki
Terada1, 2, and
Hideki
Matsui1
1 First Department of Physiology and
2 Department of Neurological Surgery, Okayama University
Medical School, Okayama 700-8558, Japan
 |
ABSTRACT |
Proteins and peptides have been demonstrated to penetrate across
the plasma membrane of eukaryotic cells by protein transduction domains. We show that protein transduction by 11 arginine
(11R) is an efficient method of delivering proteins into
the neurons of brain slices. Here, we demonstrate that PKA inhibitory
peptide, fused with 11R and nuclear localization signal,
delivers the peptide exclusively into the nuclear compartment of
neurons in brain slices. This inhibitory peptide blocked both cAMP
responsive element-binding protein phosphorylation and
long-lasting long-term potentiation (LTP) induction, but not early LTP.
These results highlight transduction of proteins and peptides into
specific neuronal subcellular compartments in brain slices as a
powerful tool for studying neuronal plasticity.
Key words:
protein transduction; poly arginine; PKA; CREB; LTP; brain slice
| |
INTRODUCTION |
A number of studies on the specific
signal transduction pathways underlying the neuronal plasticity and
electrophysiology of neurons, such as long-term potentiation (LTP) and
long-term depression (LTD), have been performed using acute brain
slices (Bliss and Collingridge, 1993 ; Bourne and Nicoll, 1993; Bear and Malenka, 1994; Steven and Sullivan, 1998). Because of its low efficiency, the expression of proteins by DNA transfection or viral
infection is limited in acute brain slices. Therefore, a method of
delivering physiologically active proteins and peptides directly into
neurons and controlling subcellular localization of these substances
within the neurons of brain slices would be advantageous in many
experiments. Recently, a human immunodeficiency virus TAT
protein transduction system has been shown to transduce biologically
active proteins into cells (Nagahara et al., 1998). The TAT protein
transduction system is capable of targeting all cell types and delivers
the proteins in rapid and concentration-dependent fashion (Schwarze et
al., 1999). Such rapid transduction is especially useful in studying
neuronal tissues such as brain slices, which deteriorate quickly.
However, the critical disadvantage of this method is its low
transduction efficiency. Here, we show that protein transduction by
means of the 11 arginines protein transduction domain (11R)
is highly efficient in delivering proteins into the neurons of brain
slices. Fusion of PKA inhibitory peptide (Kemp et al., 1988)
with 11R and nuclear localization signal (NLS) results in
the delivery of the peptide exclusively into the nuclear compartment of
neurons in brain slices. This inhibitory peptide blocked both cAMP
responsive element-binding protein (CREB) phosphorylation and
long-lasting (L)-LTP induction. This technology introduces new
opportunities to study the signal transduction systems that underlie
neuronal plasticity.
| |
MATERIALS AND METHODS |
Construction of protein transduction domain-enhanced
green fluorescence protein vectors. The
following oligonucleotides (Amersham Pharmacia Biotech, Arlington
Heights, IL) were synthesized. Oligo 11R-EGFP-S,
9R-EGFP-S, and 7R-EGFP-S correspond to the
complements of the coding strand of enhanced green fluorescence protein
(EGFP) and introduced 11, 9, and 7 arginines coding sequence and
BamHI sites. Oligo EGFP-AS corresponds to the complements of
the coding strand and introduced EcoRI site. Each mutant was
amplified by PCR using the respective primers together with the
oligonucleotides from 10 ng EGFP cDNA (Clontech, Cambridge, UK) and the
fragments subcloned into the BamHI and EcoRI
sites of pET21a (+) (Novagen, Madison, WI) using a Ligation kit
(TaKaRa, Tokyo, Japan), according to the manufacturer's instructions.
Expression and purification of recombinant proteins. BL21
(DE3) cells containing each expression plasmid were grown
at 37°C to an OD600 of 0.8. Isopropyl- -D-thiogalactopyranoside was
added to a final concentration of 0.1 mM, and
each culture was incubated for 12 hr at 24°C. Cells were harvested
and resuspended in 100 ml of lysis buffer containing 20 mM HEPES, pH8.0, 100 mM
NaCl, 8 M urea, and 20 mM
imidazol. Subsequently, the cells were sonicated, and the supernatants
were recovered and applied to a column of Ni-NTA agarose
(Invitrogen, San Diego, CA).
Synthesis of peptides. Peptides were synthesized by the
Sigma Genosis Japan. The peptides were purified by preparative
reversed-phase HPLC, were >99.8% pure as analyzed by HPLC, and had
the expected amino acid composition and mass spectra.
Protein kinase assays. PKA was obtained from Calbiochem
(LaJolla, CA). Protein kinase assays were performed by previously described methods (Matsushita and Nairn, 1998). The assay mixture was
placed on ice in a 1.5 ml Eppendorf tube that contained 3 µl of 10×
kinase buffer [500 mM HEPES, pH 7.5, 100 mM MgCl2], 3 µl of 1 mM synapsin I site 1 peptide, and each
concentration of PKI peptides. Total reaction volume was 30 µl. Assay mixtures were preincubated for 30 min, then started by
adding 3 µl of ATP mixture to make a total of 2.5 mCi of
[ -32P]ATP per tube and 50 µM ATP and allowed to progress in a 30°C water bath for 6 min. Reactions were stopped by adding 30 µl of cold
30% acetic acid, and 50 µl of each reaction mixture was applied to
P81 Whatman phosphocellulose paper (2 × 2 cm). The paper was washed four times with 250 ml of 75 mM phosphoric
acid for 10 min each time. Then, the paper was air-dried, and the
radioactivity was counted (Cerenkov counting).
Transduction of protein into Cos-7, C2C12, U251-MG, PC12, and the
primary culture of hippocampal neurons. Cos-7 cells and U251-MG
were cultured on coverslip glasses with 10% FBS. The PC12 cells were
plated on coverslips coated with
laminin-poly-D-lysine and maintained in DMEM
(Life Technologies, Gaithersburg, MD) with 10% FBS plus 5% HS.
For differentiation, PC12 cells were treated with 100 ng/ml NGF (Life
Technologies) in DMEM (Life Technologies) with 0.5% FBS. The primary
culture of hippocampal neurons was prepared by means of the previously
described modification (Yan et al., 2000). Briefly, hippocampal neurons
were dissected from 18 d rat embryos (Japan SLC Inc., Shizuoka,
Japan). The neurons were plated on coverslips coated with
laminin-poly-D-lysine and maintained in
serum-free Neurobasal medium (Life Technologies) with B27 supplements
(Life Technologies). One micromolar protein transduction domain (PTD)
proteins and peptides were incubated for 30 min, and then the medium
was changed and incubated for an additional 30 min to promote nuclear
transport of the peptide. Subsequently, cells were fixed with 4%
paraformaldehyde. FITC signals in neurons were examined using a Zeiss
confocal microscope. For immunostaining, fixed neurons were stained by
EGFP antibody (1:500; Clontech), GFAP antibody (1:200; Santa Cruz
Biotechnology, Santa Cruz, CA), and MAP-2 antibody (1:5000; Sigma, St.
Louis, MO).
Protein transduction in hippocampal slices. Hippocampal
slices from 7-week-old Wistar rats were prepared as described
previously (Lu et al., 1999a,b). Transversal slices (400 µm
thick) were incubated with 1 µM protein in
artificial CSF (ACSF) saturated with 95% O2 and
5% CO2 for 30 min at 30°C. Then, ACSF was
changed to fresh ACSF without peptides and incubated an additional 30 min to promote the nuclear transport of the peptides. After washing
with PBS three times, hippocampal slices were fixed with 4%
paraformaldehyde. For MAP-2 staining, fixed slices were incubated with
MAP-2 antibodies (1:5000; Sigma), synapsin antibody (1:200; Santa
Cruz), EGFP antibody (1:500; Clontech), and GFAP antibody (1:200; Santa
Cruz) for 24 hr. After primary antibodies were washed off,
rhodamine-conjugated secondary antibodies (1:500; Chemicon, Temecula,
CA) were incubated for 6 hr. Fluorescent images were examined using a
Zeiss confocal microscope.
Analysis of phosphorylation in brain slices. Preparation of
hippocampal slices and incubation with each peptide were performed as
described. U0126 (10 µM; Calbiochem) was
incubated with hippocampal slices for 30 min. Forskolin (10 µM; Calbiochem) and 100 µM glutamate were added 30 min after
replacement with fresh ACSF and incubated for 10 min. The
reactions were stopped by adding boiled 0.1% SDS into the slices.
Using SDS-PAGE, 10 µg of protein were separated, and the gels were
transferred to nitrocellulose filters and reacted with
anti-phospho-CREB (S133) (New England Biolabs, Beverly, MA), anti-phospho-GluR1 (S845) (Upstate Biotechnology, Lake Placid, NY), and
phospho-p44/42MAP kinase antibody (Thr202/Tyr204) (New England Biolabs).
Fluorescence measurement. To determine the fluorescence
intensity of FITC-NLS-11R-PKI and NLS-11R-EGFP
molecules in ACSF, pH 7.4, in cell free system, we used Aquacosmos
Imaging System (Hamamatsu Photonics, Shizouka, Japan). The standard
curve was determined from the intensity between
FITC-NLS-11R-PKI and NLS-11R-EGFP excited at 488 nm; the intensity showed a reasonable linear profile from 100 nM to 5 µM. On the basis
of our observation, the fluorescence intensity of
FITC-NLS-11R-PKI is 1.4-fold brighter than
NLS-11R-EGFP in ACSF at room temperature. To compare the
nucleus concentration of FITC-NLS-11R-PKI and
NLS-11R-EGFP in slices, we used the digital images of brain
slices acquired by a Zeiss confocal microscope. Analysis of signal
intensity in each 50 nucleus of slices was performed using NIH
imaging software. Then, the relative intensity of EGFP and FITC in
slices was calculated from the value obtained from cell-free analysis.
We assumed that the FITC and EGFP signals in slices would have the same
fluorescence intensity ratio with cell-free measurement, because we
measured the slices in the same pH and temperature with cell-free system.
Recording of field EPSP. The hippocampal slices were
prepared from male C57BL6 mice aged 7-8 weeks. The slices (400 µm
thick) were incubated in an interface recording chamber maintained at 28.5 ± 0.5°C for at least 1.5 hr before recording and were
constantly submerged with gas-saturated ACSF at 1.5 ml/min. The
composition of the ACSF was as follows (in mM):
NaCl, 124; KCl, 4.4; CaCl2, 2.5;
MgSO4, 1.3;
NaH2PO4, 1;
NaHCO3, 26; and glucose, 10. The intensity of the
stimulation was adjusted to produce an EPSP with a slope that was
~50% of maximum. Test stimulation was delivered once per minute
(0.017 Hz). For inducing LTP, either single or multiple trains of
stimulation at 100 Hz for 1 sec were delivered at the same intensity as
the test stimulation. The hippocampal slices were incubated with each
peptide for 45 min after stabilization of basal EPSPs. Then the slices
were stimulated at 30 min after perfusion of ACSF without peptide. Data
are shown as mean (±SEM) percentage of baseline EPSP slope.
Statistical significance was evaluated by one-way ANOVA followed by
Student's t test. The slice preparation for field recording
has been described in detail previously (Lu et al.,
1999a,b).
| |
RESULTS |
Efficient protein delivery by 11 arginines protein
transduction domain
We developed a novel, high-efficiency PTD that was based on the
TAT sequence (Frankel and Pabo, 1988; Green and Loewenstein, 1988),
which has six arginines and two lysines in the PTD sequence (Fig.
1A). On the basis of
the amino acid sequence of the PTD of other proteins, we speculated
that poly-arginine was the most important factor for membrane
penetration (Derossi et al., 1996; Elliott and O'Hare, 1997; Lindgren
et al., 2000; Rothbard et al., 2000; Schwarze and Dowdy, 2000).
Therefore, we constructed a bacteria expression vector consisting of 7 arginines (7R), 9 arginines (9R), and 11 arginines (11R) followed by EGFP (Fig.
1A). Recombinant proteins were purified under
denatured conditions and dialyzed against PBS as described previously
(Nagahara et al., 1998). To analyze the transduction ability of
arginine-based PTD-EGFP proteins, we incubated Cos-7 cells with 1 µM protein for 30 min and then analyzed by
confocal laser microscopy and immunoblotting. Without the PTD domain,
EGFP evinced no fluorescent signal in the cells. The original TAT-EGFP
showed the signal in both the cytoplasm and nucleus of Cos-7 cells. The
11R-EGFP showed a much stronger signal than the original
TAT-EGFP in all regions of the cells. Incubation with
11R-EGFP, 9R-EGFP, and 7R-EGFP
demonstrated that the arginine length is a critical factor in
determining transduction efficiency in culture cells (Fig.
1B). Immunoblotting of EGFP showed that the
efficiency of 11R in delivering the EGFP protein in Cos-7
cells was at least four times greater than that of the original TAT
domain. Reduction of the arginine length reduced the signal intensity
of EGFP in immunoblotting (Fig. 1C).
View larger version (29K):
[in this window]
[in a new window]
|
Figure 1.
Transduction of a series of PTD-EGFP proteins into
Cos-7 cells. A, Schematic representation of EGFP and
PTD-EGFPs. A series of DNAs were amplified using PCR from cDNA encoding
the EGFP (4). B, Analysis of protein transduction in
Cos-7 cells by confocal microscopy. Cos-7 cells cultured on glass
coverslips were incubated with 1 µM EGFP, TAT-EGFP,
11R-EGFP, 9R-EGFP, and
7R-EGFP. After 30 min, cells were washed three
times and incubated another 30 min and then analyzed by confocal
microscopy. Laser power was identical within each experiment. Scale
bar, 100 µm. C, The intracellular presence of the EGFP
was analyzed by Western blotting using GFP monoclonal antibody;
then, signals were analyzed by NIH Image (n = 3).
|
|
Transduction specificity of 11R domain in
different cells
Previous study demonstrated that TAT transduction domain delivered
the fusion protein into all tissues such as liver, muscle, kidney,
spleen, and brain (Schwarze et al., 1999). To examine the transduction
specificity of 11R domain in different cell lines, 11R-EGFP was incubated with C2C12 skeletal muscle cell line,
U251-MG glial cell line, and PC12 pheochromocytoma cell line.
11R-EGFP transduced efficiently into C2C12; however, weak
signals were observed in U251-MG glia cell line (Fig.
2A). We found that
11R domain did not deliver the EGFP into the PC 12 cells
(Fig. 2A) These results raise the possibility that
11R domain has the cell type specificity for protein
transduction into cells. To test this hypothesis, we induced PC12 cell
differentiation by NGF for 2 d; then we added 1 µM 11R-EGFP in cell medium. In
contrast to nondifferentiated PC12, differentiated cells acquired high
11R transduction efficiency (Fig.
2A,B). TAT protein transduction domain also showed the same transduction specificity for the
differentiated PC12 (Fig. 2C). A recent study suggests that
TAT transduction is endocytosis-dependent and mediated by the heparan
sulfate proteoglycan receptor (Tyagi et al., 2001). NGF induces
the heparan sulfate proteoglycan in PC12 cells (Katoh-Semba et
al., 1990). These results showed that 11R domain indeed had
cell type specificity to deliver the protein.
View larger version (29K):
[in this window]
[in a new window]
|
Figure 2.
Cell type specificity of 11R
protein transduction domain. A, 11R-EGFP
protein (1 µM) was incubated with cells for 30 min, after
which the medium was changed, incubated for another 30 min, and washed
three times with PBS. The cells were analyzed by confocal microscopy.
Laser power was identical within each experiment. PC12 cells were
treated with 100 ng/ml NGF (Life Technologies) in DMEM (Life
Technologies) with 0.5% FBS for 2 d. The intracellular presence
of the 11R-EGFP (B) and TAT-EGFP
(C) in PC12 cells was analyzed by Western
blotting using GFP monoclonal antibody; then, signals were analyzed by
NIH Image (n = 3). The data shown are the average
of three experiments. Three independent experiments gave similar
results.
|
|
Transduction of EGFP into the neurons of acute
hippocampal slices
Subsequently, we determined whether the 11R domain
would deliver the protein to the neurons in a brain slice. Incubation
of 11R-EGFP with brain slices for 30 min enabled delivery of
a high level of EGFP into the hippocampal neurons of the brain slice. Examination of the slices by confocal laser microscope demonstrated that 11R-EGFP was effectively introduced into the pyramidal
and granule cells in the hippocampus (Fig.
3A,B).
11R-EGFP exhibited a diffuse fluorescent signal throughout
the cytoplasm, nucleus, and dendrites. To examine how deeply into the
tissue the 11R fusion protein can penetrate hippocampal
slices, we collected serial optical sections of 12 µm steps along the
Z-dimension by confocal laser microscope. Each section in
different depths from the slice surface showed essentially the same
fluorescence intensity (Fig. 3C). Without the
11R, EGFP produced no green signal in the hippocampus (data
not shown). To examine the subcellular localization of transduction protein in neurons, we used the immunostaining studies of synapsin, a
protein present at presynapse (Greengard et al., 1993). Using immunostaining of synapsin, presynapses of mossy fibers were stained as small red dots (Fig.
4B). In contrast, green
EGFP signal was not observed in presynapses of mossy fibers (Fig.
4A). Double labeling image did not show any
colocalization with EGFP and synapsin in mossy fibers (Fig.
4C). To determine whether protein could transduce glia cells
in brain slices, slices were stained with antibody against a glia
maker, GFAP. EGFP strongly transduced into the granule cells of the
dentate gyrus (Fig. 4D), however EGFP was not
observed in GFAP-positive cells (Fig.
4E,F). These data strongly
support the idea that 11R delivered the protein into the
neurons but could not deliver into the glia cells in acute brain
slices.
View larger version (96K):
[in this window]
[in a new window]
|
Figure 3.
Transduction of 11R-EGFP into a
hippocampal brain slice. Protein (1 µM) was incubated
with the brain slice for 30 min, after which the medium was changed,
incubated for another 30 min, and washed three times with PBS. The
slices were analyzed by confocal microscopy. 11R-EGFP
was transduced in the neurons of hippocampus. EGFP signals in neurons
were examined using a Zeiss confocal microscope.
11R-EGFP was transduced into the neurons in the CA1
region (A), and CA4 and dentate gyrus
(DG) (B). Scale bars, 100 µm.
C, The images are multiple optical 12 µm step sections
spanning the Z-dimension of laser scans of the area
CA2-CA3 of hippocampal slice.
|
|
View larger version (53K):
[in this window]
[in a new window]
|
Figure 4.
Distribution of 11R-EGFP in a
hippocampal brain slice. A-C, Double
staining with EGFP (green) and
rhodamine-conjugated second antibody against synapsin antibody
(red). D-F, Double
staining with EGFP (green) and
rhodamine-conjugated second antibody against GFAP antibody
(red). MF, Mossy fiber;
DG, dentate gyrus. Scale bars:
A-C, 50 µm;
D-F, 200 µm.
|
|
Delivering the peptides and protein into the neuronal nucleus
To deliver the protein into the neuronal nucleus, we expressed
recombinant EGFP fused with 11R and the SV40 NLS.
Then, we examined whether the NLS-11R would deliver EGFP to
the nucleus of neurons in a brain slice. Examination of the brain
slices by confocal laser microscope demonstrated that EGFP was
effectively introduced into the nucleus of pyramidal cells of CA1 area
in the hippocampus (Fig. 5A).
To investigate cAMP-dependent protein kinase (PKA) function in the
neuronal nucleus, we synthesized two peptides; PKA inhibitory peptide
(PKI 10-22 amide) fused with 11R and the SV40 NLS (Fig.
5B). To evaluate the specificity of these peptides, IC50 for
PKA were measured by protein kinase assay using synapsin site I peptide
(Matsushita and Nairn, 1998). IC50 of
FITC-11R-PKI for PKA was at 92 nM, and
FITC-NLS-11R-PKI for PKA was at 103 nM. Each synthesized peptide was added to
hippocampal primary neurons for 30 min, after which the medium was
changed and incubated for another 30 min to promote nuclear transport. 11R-PKI exhibited a diffuse fluorescent signal throughout
the cytoplasm, nucleus, and cellular processes, whereas the
NLS-11R-PKI was localized specifically in the nucleus (Fig.
5C).
View larger version (42K):
[in this window]
[in a new window]
|
Figure 5.
Analysis of subcellular localization of
NLS-11R-EGFP, FITC-11R-PKI, and
FITC-NLS-11R-PKI in a hippocampal slice and primary
culture neurons. A, Transduction of
NLS-11R-EGFP into a hippocampal brain slice. Protein (1 µM) was incubated with the brain slice for 30 min, after
which the medium was changed and incubated for another 30 min. The CA1
region of slices was analyzed by confocal microscopy. B,
Schematic representation of synthesized PKA inhibitor peptides.
C, Subcellular localization of
FITC-11R-PKI and FITC-NLS-11R-PKI in
10 d primary culture neurons. Primary culture neurons were
incubated with each peptide (1 µM) for 30 min, after
which the medium was changed and incubated for another 30 min. Culture
cells were fixed by 4% paraformaldehyde and then analyzed by confocal
microscopy. Scale bars, 100 µm.
|
|
View larger version (31K):
[in this window]
[in a new window]
|
Figure 6.
Inhibition of PKA activity in brain slices.
A, Transduction of FITC-NLS-11R-PKI into
a hippocampal brain slice. Peptide (1 µM) was incubated
with the brain slice for 30 min, after which the medium was changed,
incubated for another 30 min, washed three times with PBS, and then
fixed with 4% paraformaldehyde. The fixed slices were stained
with MAP-2 monoclonal antibody. FITC signals in neurons of the brain
slices were examined using a Zeiss confocal microscope.
FITC-NLS-11R-PKI was localized in the nucleus. Pyramidal
neurons in CA1 were detected by MAP-2 antibody (red).
The nuclei of pyramidal neurons showed the green
FITC-NLS-11R-PKI signal. Scale bar, 50 µm.
B, Reduced phosphorylation of CREB at serine 133 by
11R-PKI and NLS-11R-PKI. Hippocampal
brain slices were incubated at the indicated peptide concentration and
control (absence of peptide) for 1 hr, after which the ACSF was
changed and incubated 30 min. The slices were stimulated by
10 µM of forskolin (Forsk) for 10 min. The
reactions were immediately stopped by adding 0.1% SDS into the slices.
Then, samples were analyzed by Western blotting using phospho-Ser133
CREB specific antibody, phospho-Ser 845 GluR1 antibody, and actin
antibody (n = 3). C,
11R-PKI did not inhibit the MAP kinase cascade.
Hippocampal brain slices were incubated at the indicated peptide
concentration, control (Cont; absence of peptide), and
10 µM U0126 for 30 min, after which the ACSF was changed
and incubated 30 min. The slices were stimulated by 100 µM glutamate (Glu) for 10 min. Samples
were analyzed by Western blotting using phospho-p42/44 MAP kinase
antibody and actin antibody (n = 3). Three
independent experiments gave similar results.
|
|
Next, we determined whether the NLS-11R would deliver PKI
peptide to the nucleus of neurons in a brain slice. Incubation of NLS-11R-PKI with brain slices for 30 min enabled delivery of
a high level of peptides into the neurons of brain slices. Examination of the brain slices by confocal laser microscope demonstrated that
NLS-11R-PKI peptide was effectively introduced into the
nucleus of pyramidal cells in the hippocampus (Fig.
6A). Double staining of
MAP-2 and NLS-11R-PKI in the CA1 region showed that the
inhibitory peptide was localized exclusively in the nuclear compartment
of pyramidal neurons (Fig. 6A).
To determine whether the NLS-11R-PKI peptide was
physiologically active as a PKA inhibitor after transduction into brain
slices, we examined the phosphorylation of Ser133 of the CREB and
Ser845 of the GluR1. It has been show that Ser133 of the CREB was
phosphorylated by multiple protein kinases such as PKA,
calmodulin-dependent protein kinase IV, extracellular signal-regulated
protein kinases (ERK), and Rsk2 in the nucleus (Gonzalez and Montminy,
1989; Bito et al., 1996; Xing et al., 1996). A recent
study showed that Ser845 of the GluR1 was phosphorylated specifically
by PKA in the postsynaptic regions (Kameyama et al., 1998).
Phospho-specific antibodies for each site were used to examine the
change of phosphorylation level of each site through PKA inhibition by
the transduction of inhibitory peptides in brain slices. Even 100 nM NLS-11R-PKI almost completely blocked the phosphorylation of CREB by PKA, which was stimulated by
forskolin. Phosphorylation of CREB was inhibited partially by 100 nM 11R-PKI and completely by 1 µM. In contrast, NLS-11R-PKI did not
reduce the Ser 845 of the GluR1, even at a concentration of 1 µM. However, 11R-PKI significantly
reduced the phosphorylation of the GluR1 at a concentration of 1 µM (Fig. 6B). These results suggested that NLS-11R-PKI specifically blocked the PKA
activity in the nucleus, but not in the synaptic regions. We next
determined whether mitogen-activated protein kinase (MAPK)/(ERK) kinase
(MEK) was inhibited when the slices were stimulated by glutamate in the
presence of the 11R-PKI and U0126, a specific inhibitor of MEK1/2. Stimulation of metabotropic glutamate receptors leads to an
activation of the MAPK and ERK (English and Sweatt,
1996; Ferraguti et al., 1999). As shown in Figure 6C,
application of 10 µM U0126 reduced the
phosphorylation of p42 and p44 MAPK. In contrast, 11R-PKI
had no effect on the phosphorylation of MAPK by MEK. These data showed
that PKI peptides were specific for PKA but not for MAP kinase cascade.
NLS-11R-PKI blocks L-LTP induction, but not
early LTP
LTP is activity-dependent strengthening of synaptic efficacy;
early (E)-LTP can last for 1 hr, and L-LTP can last >3 hr, depending on the stimulation pattern (Frey et al., 1993; Huang and Kandel, 1994).
Phosphorylation of CREB by PKA is thought to be a critical step
for induction of L-LTP in the CA1 region of the hippocampus (Frey et
al., 1993; Huang and Kandel, 1994; Abel et al., 1997; Impey et al.,
1998). PKA acts in multiple subcellular compartments and phosphorylates
different substrates such as AMPA receptor, inhibitor-1, and CREB in
neurons (Gonzalez and Montminy, 1989; Shenolikar and Nairn,
1991; Kameyama et al., 1998). Conventional pharmacological studies have
failed to answer the critical questions of whether PKA phosphorylates
the substrates for induction of L-LTP in the nucleus or in the synaptic
regions in neurons.
We examined the effect of NLS-11R-PKI on E-LTP
and L-LTP in the CA1 region of the mouse hippocampus.
NLS-11R-PKI did not impair basic electrophysiological
activity such as basal EPSP and paired pulse facilitation (Table
1). Moreover, NLS-11R-PKI peptide did not
block the E-LTP elicited by a single train in brain slices (Fig.
7A). EPSP amplitude in
NLS-11R-PKI peptide-incubated slices was the same as that of
the control and NLS-11R-EGFP-treated slices. We also
determined the relative fluorescence intensity between
FITC-11R-NLS-PKI and NLS-11R-EGFP signals in
these slices. The fluorescence signals of both molecules were of the
same intensity in the nucleus of these slices as described as Materials
and Methods. Therefore, we assumed the concentration of
NLS-11R-PKI and NLS-11R-EGFP in the nucleus was
almost the same in these slices. Three trains of 100 Hz for 1 sec
stimulation induced stable long-lasting LTP (the EPSP slope was
164 ± 12.6% of baseline 3 hr after the end of tetanization;
n = 6) (Fig. 7B). NLS-11R-EGFP
delivered the EGFP into the nucleus of hippocampus (Fig.
5A) and had no effect on L-LTP (158 ± 10.6%;
n = 6). In contrast, NLS-11R-PKI
significantly inhibited L-LTP 3 hr after stimulation (121 ± 11%;
n = 6; F(1,30) = 31.56; p < 0.01 compared with control) (Figure
7B,C). These data suggest that
inhibition of PKA activity in the nucleus was sufficient to prevent the
induction of L-LTP in the CA1 of the hippocampus.
View this table:
[in this window]
[in a new window]
|
Table 1.
Comparison of paired-pulse facilitation (PPF) and field
EPSP (fEPSP) slope in NLS-11R-GFP- and
NLS-11R-PKI-treated slices and controls
|
|
View larger version (27K):
[in this window]
[in a new window]
|
Figure 7.
NLS-11R-PKI peptides inhibited the
L-LTP induction, but not that of E-LTP. A,
NLS-11R-PKI did not block E-LTP induced by one train of
100 Hz for 1 sec tetanization (arrow). B,
NLS-11R-PKI significantly inhibited L-LTP induced by
three trains of 100 Hz for 1 sec tetanization (arrows).
In contrast, NLS-11R-GFP had no effect on the L-LTP.
Insets, Representative field EPSPs before and 3 hr after
tetanic stimulations are shown. Calibration: 10 msec, 2 mV.
C, Comparisons of EPSPs slope 1 hr
(a) and 3 hr (b) after
tetanic stimulation for induction of either E-LTP or L-LTP,
respectively. *p < 0.01 compared with the control
slices.
|
|
| |
DISCUSSION |
It has been demonstrated that proteins and peptides penetrate
across the plasma membrane of eukaryotic cells by means of protein transduction domains (Lindgren et al., 2000; Schwarze and Dowdy, 2000).
Protein transduction by 11R was highly efficient in
delivering proteins into culture cells and brain slices, especially
into the neurons. The mechanism of plasma membrane transduction by 11R is still to be elucidated; however, recent study of TAT
transduction system suggests that transduction is mediated by
endocytosis mediated by heparan sulfate proteoglycan receptors. In
contrast, oligopeptide penetratin derived from the homeodomain of
Antennapedia has shown to translocate across pure lipid bilayers
(Thoren et al., 2000). 11R domain requires the
differentiation by NGF treatment for plasma membrane transduction in
PC12 cells (Fig. 2). The results support the idea that the uptake
mechanism involves receptor or transporter-dependent pathway.
Therefore, expression level of receptors for 11R on the cell
membrane might be a critical factor for cell type specificity of
protein transduction.
The results of our studies using brain slices are sufficient to show
how effective this system is for analyzing the molecular mechanism in
neurons. Previous studies suggest that PKA plays a key role in
induction of L-LTP through the phosphorylation of CREB in the
hippocampus (Abel et al., 1997; Impey et al., 1998). However, it is
difficult to show whether PKA acts in the nucleus or in other
compartments of the neurons during the induction of L-LTP.
NLS-11R-PKI showed that inhibition of PKA in the nucleus decreased the phosphorylation of Ser133-CREB and blocked L-LTP induction. The data indicated that for induction of L-LTP, active PKA
translocation into the nucleus is essential. These results agreed very
well with the data from hippocampus-specific PKA regulatory subunit
transgenic mice (Abel et al., 1997). In this transgenic experiment,
however, PKA activity was inhibited both at the synapses and in the
cytoplasm by overexpression of the regulatory subunit of PKA. Our data
define the role of PKA in the nucleus during L-LTP induction in the CA1 region.
Genetic and pharmacological studies have provided important information
regarding the molecular function of neural physiology. In addition to
these methods, the protein transduction system provides many advantages
for analysis of molecular function and the electrophysiology of neurons
as described in this report. To carry the molecular
analysis of signal transduction and electrophysiology in neurons
further, it will be necessary to develop the delivery of proteins,
compounds, antisense oligonucleotides, etc., to neuronal subcellular
compartments such as the nucleus, postsynaptic terminal, and
presynaptic terminal in brain slices.
| |
FOOTNOTES |
Received Feb. 13, 2001; revised May 17, 2001; accepted May 21, 2001.
This work was supported by a Grant-in-Aid for Scientific Research on
Priority Areas (C) from the Ministry of Education, Science, Sports and
Culture of Japan. We thank T. Takahashi for discussion and comments on
this manuscript.
Correspondence should be addressed to Hideki Matsui, First Department
of Physiology, Okayama University Medical School, 2-5-1 Shikata-cho,
Okayama 700-8558, Japan. E-mail:
matsuihi{at}cc.okayama-u.ac.jp.
| |
REFERENCES |
-
Abel T,
Nguyen PV,
Barad M,
Deuel TA,
Kandel ER,
Bourtchouladze R
(1997)
Genetic demonstration of a role for PKA in late phase of LTP and hippocampus-based long-term memory.
Cell
88:615-626[ISI][Medline].
-
Bear MF,
Malenka RC
(1994)
Synaptic plasticity: LTP and LTD.
Curr Opin Neurobiol
4:389-399[Medline].
-
Bito H,
Deisseroth K,
Tsien RW
(1996)
CREB phosphorylation and dephosphorylation: a Ca2+ and stimulus duration-dependent switch for hippocampal gene expression.
Cell
87:1203-1214[ISI][Medline].
-
Bliss TV,
Collingridge GL
(1993)
A synaptic model of memory: long-term potentiation in the hippocampus.
Nature
361:31-39[Medline].
-
Bourne HR,
Nicoll R
(1993)
Molecular machines integrate coincident synaptic signal.
Cell
72:65-75.
-
Derossi D,
Calvet S,
Trembleau A,
Brunissen A,
Chassaing G,
Prochiantz A
(1996)
Cell internalization of the third helix of Antennapedia homeodomain is receptor-independent.
J Biol Chem
271:18188-18193.6[Abstract/Free Full Text].
-
Elliott G,
O'Hare P
(1997)
Intracellular trafficking and protein delivery by a herpes virus structure protein.
Cell
88:223-233[ISI][Medline].
-
English JD,
Sweatt JD
(1996)
Activation of p42 mitogen-activated protein kinase in hippocampal long-term potentiation.
J Biol Chem
271:24329-24332[Abstract/Free Full Text].
-
Ferraguti F,
Baldani-Guerra B,
Corsi M,
Nakanishi S,
Corti C
(1999)
Activation of the extracellular signal-regulated kinase 2 by metabotropic glutamate receptors.
Eur J Neurosci
11:2073-2082[ISI][Medline].
-
Frankel AD,
Pabo CO
(1988)
Cellular uptake of the tat protein from human immunodeficiency virus.
Cell
55:1189-1193[ISI][Medline].
-
Frey U,
Huang YY,
Kandel ER
(1993)
Effects of cAMP simulate a late stage of LTP in hippocampal CA1 neurons.
Science
260:1661-1664[Abstract/Free Full Text].
-
Gonzalez GA,
Montminy MR
(1989)
Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.
Cell
59:675-680[ISI][Medline].
-
Green G,
Loewenstein PM
(1988)
Autonoumous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein.
Cell
55:1179-1188[ISI][Medline].
-
Greengard P,
Valtorta F,
Czernik AJ,
Benfenati F
(1993)
Synaptic vesicle phosphoproteins and regulation of synaptic function.
Science
259:780-785[Abstract/Free Full Text].
-
Huang YY,
Kandel ER
(1994)
Recruitment of long-lasting and protein kinase A-dependent long-term potentiation in the CA1 region of hippocampus requires repeated tetanization.
Learn Mem
1:74-82[Abstract/Free Full Text].
-
Impey S,
Obrietan K,
Wong ST,
Poser S,
Yano S,
Wayman G,
Deloulme JC,
Chan G,
Storm DR
(1998)
Cross talk between ERK and PKA is required for Ca2+ stimulation of CREB-dependent transcription and ERK nuclear translocation.
Neuron
21:869-883[ISI][Medline].
-
Kameyama K,
Lee HK,
Bear MF,
Huganir RL
(1998)
Involvement of a postsynaptic protein kinase A substrate in the expression of homosynaptic long-term depression.
Neuron
21:1163-1175[ISI][Medline].
-
Katoh-Semba R,
Oohira A,
Kashiwamata S
(1990)
Changes in glycosaminoglycans during the neuritogenesis in PC12 pheochromocytoma cells induced by nerve growth factor.
J Neurochem
55:1749-1757[Medline].
-
Kemp BE,
Cheng HC,
Walsh DA
(1988)
Peptide inhibitors of cAMP-dependent protein kinase.
Methods Enzymol
159:173-183[ISI][Medline].
-
Lindgren M,
Hällbrink M,
Prochiantz A,
Langel Ü
(2000)
Cell-penetrating peptides.
Trends Pharmacol Sci
21:99-103[Medline].
-
Lu YF,
Kandel ER,
Hawkins RD
(1999a)
Nitric oxide signaling contributes to late-phase LTP and CREB phosphorylation in the hippocampus.
J Neurosci
19:10250-10261[Abstract/Free Full Text].
-
Lu YF,
Kojima N,
Tomizawa K,
Moriwaki A,
Matsushita M,
Obata K,
Matsui H
(1999b)
Enhanced synaptic transmission and reduced threshold for LTP induction in fyn-transgenic mice.
Eur J Neurosci
11:75-82[ISI][Medline].
-
Matsushita M,
Nairn AC
(1998)
Characterization of the mechanism of regulation of Ca2+/calmodulin-dependent protein kinase I by calmodulin and by Ca2+/calmodulin-dependent protein kinase kinase.
J Biol Chem
273:21473-21481[Abstract/Free Full Text].
-
Nagahara H,
Vocero-Akbani AM,
Snyder EL,
Ho A,
Latham DG,
Lissy NA,
Becker-Hapak M,
Ezhevsky SA,
Dowdy SF
(1998)
Transduction of full-length TAT fusion proteins into mammalian cells: TAT-p-27Kip1 induces cell migration.
Nat Med
4:1449-1452[ISI][Medline].
-
Rothbard JB,
Garlington S,
Lin Q,
Kirschberg T,
Kreider E,
McGrane PL,
Wender PA,
Khavari PA
(2000)
Conjugation of arginine oligomers to cyclosporin A facilitates topical delivery and inhibition of inflammation.
Nat Med
6:1253-1257[ISI][Medline].
-
Schwarze S,
Dowdy SF
(2000)
In vivo protein transduction: intracellular delivery of biologically active proteins, compounds and DNA.
Trends Pharmacol Sci
21:45-48[Medline].
-
Schwarze SR,
Ho A,
Vocero-Akbani AM,
Dowdy SF
(1999)
In vivo protein transduction: delivery of a biologically active protein into the mouse.
Science
285:1569-1572[Abstract/Free Full Text].
-
Shenolikar S,
Nairn AC
(1991)
Protein phosphatase: recent progress.
Adv Second Messenger Phosphoprotein Res
23:1-121[ISI][Medline].
-
Steven CF,
Sullivan J
(1998)
Synaptic plasticity.
Curr Biol
8:151-153.
-
Thoren PE,
Persson D,
Karlsson M,
Norden B
(2000)
The antennapedia peptide penetratin translocates across lipid bilayers: the first direct observation.
FEBS Lett
482:265-268[Medline].
-
Tyagi M,
Rusnati M,
Presta M,
Giacca M
(2001)
Internalization of HIV-1 Tat requires cell surface heparan sulfate proteoglycans.
J Biol Chem
276:3254-3261[Abstract/Free Full Text].
-
Xing J,
Ginty DD,
Greenberg ME
(1996)
Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase.
Science
273:959-963[Abstract].
-
Yan Z,
Hsieh-Wilson L,
Feng J,
Tomizawa K,
Allen PB,
Fienberg AA,
Nairn AC,
Greengard P
(2000)
Protein phosphatase 1 modulation of neostriatal AMPA channels: regulation by DARPP-32 and spinophilin.
Nat Neurosci
2:13-17.
Copyright © 2001 Society for Neuroscience 0270-6474/01/21166000-08$05.00/0
This article has been cited by other articles:
|
|
|
|
 
C. P. Bailey, R. E. Nicholls, X.-l. Zhang, Z.-y. Zhou, W. Muller, E. R. Kandel, and P. K. Stanton
G{alpha}i2 inhibition of adenylate cyclase regulates presynaptic activity and unmasks cGMP-dependent long-term depression at Schaffer collateral-CA1 hippocampal synapses
Learn. Mem.,
April 7, 2008;
15(4):
261 - 270.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Ogawa, S. Ono, T. Ichikawa, S. Arimitsu, K. Onoda, K. Tokunaga, K. Sugiu, K. Tomizawa, H. Matsui, and I. Date
Novel Protein Transduction Method by Using 11R: An Effective New Drug Delivery System for the Treatment of Cerebrovascular Diseases
Stroke,
April 1, 2007;
38(4):
1354 - 1361.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. A. Kovacs, P. Steullet, M. Steinmann, K. Q. Do, P. J. Magistretti, O. Halfon, and J.-R. Cardinaux
TORC1 is a calcium- and cAMP-sensitive coincidence detector involved in hippocampal long-term synaptic plasticity
PNAS,
March 13, 2007;
104(11):
4700 - 4705.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
P. V. Nguyen
Comparative plasticity of brain synapses in inbred mouse strains
J. Exp. Biol.,
June 15, 2006;
209(12):
2293 - 2303.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Song, P. Smolen, E. Av-Ron, D. A. Baxter, and J. H. Byrne
Bifurcation and Singularity Analysis of a Molecular Network for the Induction of Long-Term Memory
Biophys. J.,
April 1, 2006;
90(7):
2309 - 2325.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Noguchi, S. Bonner-Weir, F.-Y. Wei, M. Matsushita, and S. Matsumoto
BETA2/NeuroD Protein Can Be Transduced Into Cells Due to an Arginine- and Lysine-Rich Sequence
Diabetes,
October 1, 2005;
54(10):
2859 - 2866.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. C. Warburton, C. P. J. Glover, P. V. Massey, H. Wan, B. Johnson, A. Bienemann, U. Deuschle, J. N. C. Kew, J. P. Aggleton, Z. I. Bashir, et al.
cAMP Responsive Element-Binding Protein Phosphorylation Is Necessary for Perirhinal Long-Term Potentiation and Recognition Memory
J. Neurosci.,
July 6, 2005;
25(27):
6296 - 6303.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sakaguchi, T. Nukui, H. Sonegawa, H. Murata, J. Futami, H. Yamada, and N.-h. Huh
Targeted disruption of transcriptional regulatory function of p53 by a novel efficient method for introducing a decoy oligonucleotide into nuclei
Nucleic Acids Res.,
May 26, 2005;
33(9):
e88 - e88.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Michiue, K. Tomizawa, F.-Y. Wei, M. Matsushita, Y.-F. Lu, T. Ichikawa, T. Tamiya, I. Date, and H. Matsui
The NH2 Terminus of Influenza Virus Hemagglutinin-2 Subunit Peptides Enhances the Antitumor Potency of Polyarginine-mediated p53 Protein Transduction
J. Biol. Chem.,
March 4, 2005;
280(9):
8285 - 8289.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Tomizawa, S. Sunada, Y.-F. Lu, Y. Oda, M. Kinuta, T. Ohshima, T. Saito, F.-Y. Wei, M. Matsushita, S.-T. Li, et al.
Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles
J. Cell Biol.,
November 24, 2003;
163(4):
813 - 824.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Noguchi, H. Kaneto, G. C. Weir, and S. Bonner-Weir
PDX-1 Protein Containing Its Own Antennapedia-Like Protein Transduction Domain Can Transduce Pancreatic Duct and Islet Cells
Diabetes,
July 1, 2003;
52(7):
1732 - 1737.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. H. Woo and P. V. Nguyen
Protein Synthesis Is Required for Synaptic Immunity to Depotentiation
J. Neurosci.,
February 15, 2003;
23(4):
1125 - 1132.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. N. Duffy and P. V. Nguyen
Postsynaptic Application of a Peptide Inhibitor of cAMP-Dependent Protein Kinase Blocks Expression of Long-Lasting Synaptic Potentiation in Hippocampal Neurons
J. Neurosci.,
February 15, 2003;
23(4):
1142 - 1150.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Takenobu, K. Tomizawa, M. Matsushita, S.-T. Li, A. Moriwaki, Y.-F. Lu, and H. Matsui
Development of p53 Protein Transduction Therapy Using Membrane-permeable Peptides and the Application to Oral Cancer Cells
Mol. Cancer Ther.,
October 1, 2002;
1(12):
1043 - 1049.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. C. Mai, H. Shen, S. C. Watkins, T. Cheng, and P. D. Robbins
Efficiency of Protein Transduction Is Cell Type-dependent and Is Enhanced by Dextran Sulfate
J. Biol. Chem.,
August 9, 2002;
277(33):
30208 - 30218.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Peitz, K. Pfannkuche, K. Rajewsky, and F. Edenhofer
Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes
PNAS,
April 2, 2002;
99(7):
4489 - 4494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Peitz, K. Pfannkuche, K. Rajewsky, and F. Edenhofer
Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: A tool for efficient genetic engineering of mammalian genomes
PNAS,
April 2, 2002;
99(7):
4489 - 4494.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
TOP
ABSTRACT
INTRODUCTION
