From the Institut für Neurobiochemie, Universität Witten/Herdecke, Stockumer Straße 10, D-58448 Witten, Germany
Received for publication, October 10, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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The mechanism by which signals such as those
produced by glutamate are transferred to the nucleus may involve direct
transport of an activated transcription factor to trigger long-term
transcriptional changes. Ionotropic glutamate receptor
activation or depolarization activates transcription factor NF- For short-term signals (e.g. synaptic activity) to
trigger long-term changes, differential gene expression is required
(1-4). This raises the issue of determining the signaling systems that translate short-term signals to changes in gene expression. Two possible mechanisms are a signal transducer that is
retrograde-transported and that subsequently transmits information to a
transcription factor, or a transcription factor that independently can
fulfill both functions. There is evidence for each mechanism in
different systems. With regard to the former mechanism, the nerve
growth factor TrkA receptor functions in complex with nerve growth
factor as a retrograde signal transducer, connecting extracellular
signals at distant sites with nuclear gene expression via
phosphorylation of the transcription factor
CREB1 (5). NF- To date, five mammalian NF- In this study, we examined whether activated NF- Cell Culture--
Hippocampal neurons were cultured from
embryonic day 17 or 18 rats as described by Banker and Cowan (32) and
detailed by de Hoop et al. (33). One day prior to
preparation of the hippocampal neurons, neurobasal medium (Life
Technologies, Inc.) supplemented with B27 (1:50; Life Technologies,
Inc.) and 0.5 mM L-glutamine was conditioned by
astrocyte co-culture. Dissected hippocampi were treated with trypsin (2 mg/ml; Sigma) in Hanks' balanced saline solution without calcium or
magnesium and then with soybean trypsin inhibitor (1 mg/ml). Neurons
were dissociated by strokes with a fire-polished Pasteur pipette and
were suspended in minimal essential medium with Earle's salts
containing 1 mM pyruvate, 26 mM
Na2HCO3, 2 mM
L-glutamine, 20 mM KCl, and 10%
heat-inactivated horse serum. Cells (2 × 104
cells/cm2) were plated on glass coverslips coated with
polyethyleneimine (1:1000 in borate buffer, pH 7.2; Sigma). After
3 h, the cells adhered and were moved to astrocyte co-cultures
with conditioned neurobasal medium. Biolistic experiments were
performed after neurons had been maintained in culture for at least 7 days. At this age, the cultures contain mainly
N-methyl-D-aspartic acid type synapses
clustered in varicosities (34).
HEK 293 cells (American Type Culture Collection) were seeded on glass
coverslips (104 cells/cm2) in Dulbecco's
modified Eagle's medium supplemented with 10% fetal calf serum, 100 unit/ml penicillin, and 0.1 mg/ml streptomycin. The following day,
calcium phosphate transfection was performed as described previously
(26); and 24 h later, cells were fixed with 4% paraformaldehyde.
Luciferase assays were performed as described previously (14), and
standardization was performed using Renilla reniformis
luciferase together with Photinus pyralis luciferase
(Dual-Luciferase, Promega, Mannheim, Germany) as suggested by the manufacturer.
Immunostaining--
On day 8 in culture, hippocampal neurons
were treated with either 100 or 500 µM glutamate or 100 µM kainate in glia-conditioned medium for 5 min. After
two washing steps with neurobasal medium, cells were incubated at
37 °C for 1, 1.5, or 2 h. The cells were then fixed for 2 min
in ethanol and for 5 min in 3.7% formaldehyde. After washing with
phosphate-buffered saline, the cells were incubated in 5% goat serum
for 30 min at room temperature, followed by two 5-min washes with
phosphate-buffered saline. Cells were then incubated with anti-RelA
monoclonal antibody (1:50 dilution; Roche Molecular Biochemicals,
Mannheim). Anti-RelA antibody was detected by Cy3-conjugated anti-mouse
IgG (1:400 dilution; Dianova, Hamburg, Germany); staining was performed
subsequently; and detection was finished when incubation with the next
primary antibody was started. Cells incubated with secondary
antibodies, but without primary antibodies, were used as a control. The
state of RelA activation in these cultures was tested using the
anti-RelA monoclonal antibody because this antibody is commercially
available and is specific for the activated form of the RelA subunit
(26). Immunoreactivity with this antibody is detectable only after the
activation of NF- Plasmids--
A pcDNA3 expression vector (Invitrogen, Leek,
Netherlands), a pEGFP-1 vector (CLONTECH, Palo
Alto, CA), a cytomegalovirus-driven human RelA expression vector (37),
and a cytomegalovirus-driven human I Protein Extracts--
HEK 293 cells were grown on 10-cm culture
dishes; and 1 day after calcium phosphate precipitation, the
expression of EGFP-RelA and EGFP-(NLSmut)RelA was detected. Cells were
treated with buffer containing 20 mM HEPES, pH 7.9, 350 mM NaCl, 20% glycerol, 1% Nonidet P-40, 1 mM
MgCl2, 0.5 mM EDTA, 0.1 mM EGTA,
0.5 mM dithiothreitol, 2 mM
phenylmethylsulfonyl fluoride, and 0.1% aprotinin, and the protein
concentration was determined using Bradford dye reagent. Extracts were
frozen in liquid nitrogen and stored at Western Blotting--
Extracts containing 25 µg of protein
were boiled for 5 min in sample buffer and separated on an SDS-gradient
polyacrylamide minigel (4, 8, and 12%; Novel, Heidelberg, Germany).
Proteins were transferred at 1.5 mA/cm2 for 1 h onto
polyvinylidene difluoride membrane (Roche Molecular Biochemicals).
Chromogenic detection of alkaline phosphatase-labeled antibodies was
performed with a chromogenic Western Blotting kit (Roche Molecular
Biochemicals) as recommended by the manufacturer. Nonspecific binding
was blocked using 1% blocking solution for 30 min, and the blot was
washed twice with 2× buffer containing 50 mM Tris base,
150 mM NaCl, and 0.1% (v/v) Tween 20, pH 7.5, for 10 min.
Primary antibodies to GFP (0.4 µg/ml) and RelA (26) were used (both
from Roche Molecular Biochemicals).
Electrophoretic Mobility Shift Assay--
Electrophoretic
mobility shift assay was performed as described previously (6).
Oligonucleotides (Promega) encompassing the NF- Biolistic Transfection of Neurons--
Gold particles were
coated, and cartridges were prepared as described by the manufacturer
(Bio-Rad) with slight modifications (39). Briefly, 30 µg of gold (1 µm in diameter) was suspended in 60 µl of 0.05 M
spermidine (Sigma, Deisenhofen, Germany), and 60 µl of DNA (0.5 µg/µl) was added. DNA was precipitated on the particles using 60 µl of 2 M CaCl2. DNA-coated particles were suspended in 4 ml of a polyvinylpyrrolidone solution (0.05 mg/ml absolute ethanol; Sigma). Cartridges were prepared for use with the
Helios Gene Gun (Bio-Rad) with a helium pressure of 100 p.s.i.
Fluorescence Imaging of Live Cells--
Hippocampal neurons were
imaged 24 h after biolistic transfection at 30 °C in
custom-built observation chambers (courtesy of Prof. Dr. Rainer
Greger) using an Axiovert 100 microscope (Carl Zeiss, Jena,
Germany), high numerical aperture, oil immersion lenses, and a
fluorescein isothiocyanate filter set (Carl Zeiss). Images were
captured using a 2-s exposure every 20 min on Eastman Kodak 3200 ASA
color slide films using light from a 50-watt mercury lamp (HBO50, Carl
Zeiss). Care was taken to minimize exposure to the light. Cellular
photodamage was prevented by perfusion via gravity feed with
Subcellular Distribution and Biochemical Analysis of NF-
The EGFP-RelA fusion protein was found exclusively in the nucleus (Fig.
1B), whereas unfused EGFP was randomly distributed across
all cellular compartments, including the nucleus and cytoplasm (Fig.
1B). Overexpression of I
To biochemically characterize the fusion proteins, transfected HEK 293 cell extracts were separated on SDS gels and analyzed using Western
blotting (Fig. 2A). A band
corresponding to the predicted protein molecular mass was detected with
antibodies directed against either the RelA portion (Fig.
2A, lane 5) or the EGFP portion (lane
1) of the fusion protein. A second, faster migrating band was
present that might be a degradation product. The specificity of the
immunolabeling was tested via the expression of EGFP (Fig.
2A, lane 3), which was recognized only by the
antibody to GFP and not by the antibody to RelA (lane 7).
Similarly, overexpressed RelA was not detected by the antibody to GFP
(Fig. 2A, lane 2), but was detected by the
antibody to RelA (lane 6). Mock-transfected cells did not
react specifically with either antiserum (Fig. 2A, lanes 4 and 8).
The DNA-binding characteristics of the EGFP-RelA fusion protein were
analyzed by electrophoretic mobility shift assay (Fig. 2B).
Using a
The transcriptional capability of the EGFP-RelA fusion protein was
analyzed using an NF- Characterization of Mechanisms of Transport into the
Nucleus--
We constructed an EGFP-RelA fusion protein with a
mutated NLS containing three point mutations (38) (Fig.
4A). To study the mechanisms
of transport into the nucleus of hippocampal neurons (Fig.
4B, left panels) or HEK 293 cells (right
panels), cells were transfected as indicated. EGFP-RelA was
localized primarily in the nucleus (Fig. 4B), whereas
EGFP-RelA with a mutated NLS was localized primarily in the cytoplasm,
suggesting that an intact NLS is essential for a nuclear distribution
of EGFP-RelA. Low levels of EGFP-RelA with a mutated NLS were detected
in the nucleus, which might be due to additional auxiliary nuclear
localization sequences (28). This effect was observed in both
hippocampal neurons and HEK 293 cells, suggesting that the distribution
of NF- Coexpression of I Activation of Endogenous RelA via Glutamate and Kainate--
We
determined whether endogenous NF- Redistribution of EGFP-RelA after Depolarization--
To
investigate the stimulus-dependent transport of EGFP-RelA
from neurites to the nucleus, cultures were treated with 100 mM KCl for 5 min (Fig. 7).
Note that under control conditions, EGFP-RelA was localized in long
neurites and in puncta resembling varicosities (Fig. 7, upper
panel). Ninety min after treatment with KCl, EGFP-RelA was
redistributed from neurites and the soma to the nucleus. Thus, we
wished to test other stimuli for the capability to induce NF- Time-dependent Redistribution of EGFP-RelA--
To
follow the transport of EGFP-RelA from neurites to the nucleus over
time, cultures were treated with 500 µM glutamate after an adaptation time of several minutes following transfer to the imaging
chamber (Fig. 8A). After
treatment, a gradual transport of EGFP-RelA from neuronal processes to
the nucleus was observed (Fig. 8A, panels a-f).
By 80 min post-treatment (Fig. 8A, panel e), the
EGFP-RelA fusion protein filled the nucleus, consistent with fast
retrograde transport. A stimulus-dependent change in the
intensity of the EGFP-RelA fluorescence along a thin line through the
center of a neuron (Fig. 8A, panel a) is shown
Fig. 8B. It is evident that a fraction of EGFP-RelA was
sharply redistributed from neurites to the nucleus, suggesting
retrograde transport.
Similar transport of EGFP-RelA was observed in cultures stimulated with
100 µM kainate (see below). In contrast to the EGFP-RelA results, the EGFP-RelA fusion protein with mutated NLS was not transported from neuronal processes to the nucleus after either glutamate (Fig. 8C) or kainate (see below) stimulation.
Similar results were obtained from five cells recorded on different days.
For better visualization and comparison with controls, pseudo-color
images were created from the digitally captured images in Fig. 8
(A, panels a and f; and C,
panels a and c) (Fig.
9). Interestingly, EGFP-RelA was present
in puncta resembling varicosities under unstimulated conditions (Fig.
9a), but it was not present in puncta after stimulation with
glutamate (Fig. 9b), when also the neurites were free of
activated NF-
For this purpose, we randomly selected five neurons from EGFP-RelA
experiments or from independent EGFP-(NLSmut)RelA experiments. From
these neurons, the GFP fluorescence was measured in several neurites
before (I0) and after treatment (I)
with glutamate or kainate along a thin line (as described for Fig.
8B). The I/I0 × 100 ratio
was depicted as a measure of GFP transport. The strong decrease in
fluorescence intensity in neurites from EGFP-RelA-transfected neurons
demonstrated the retrograde transport of activated NF- To enable us to analyze the translocation of GFP-tagged RelA in
living hippocampal neurons, we fused a GFP tag to the RelA subunit of
NF- We found, in untreated hippocampal neurons cultured as astrocyte
co-cultures, endogenous RelA immunoreactivity in neuronal processes,
the soma, and varicosities, whereas no immunoreactivity was found in
the nucleus. No constitutive NF- The results of this study indicate that NF- The capability of NF- To investigate mechanisms involved in the retrograde transport of
proteins in neurons, Ambron and coworkers (48, 49) analyzed protein
transport from the soma to the axon and vice versa in Aplysia
californica. They found that both the retrograde transport of
proteins from synapses to the nucleus and the transport of proteins
into the nucleus are dependent on an NLS derived from the SV40 large
T-antigen (49-51). Interestingly, RelA contains an NLS that is very
similar to the SV40-derived sequence that functions in
Aplysia. Homologous NLS motifs are located within the basic
region of the DNA-binding domain of the activating transcription factor
family of transcriptions factors. Recently, immunostaining and Western
blot analysis revealed retrograde transport of the activating
transcription factor in nociceptive neurons (52). Here, we found that
in mammalian neurons, the retrograde transport of EGFP-RelA was
dependent on a functional NLS, supporting the idea of an evolutionarily
conserved transporting machinery that might be used by different
transcription factors. After I Transport of transcription factors from synapses to the nucleus,
e.g. in response to specific stimuli, might be a regulating mechanism of gene expression. In this study, the EGFP-tagged RelA subunit of NF- In accordance with biochemical data (38) and crystal structure studies
(28), the results of this study indicate that nuclear translocation of
NF-B and
leads to translocation of NF-
B from the cytoplasm to the nucleus. We
investigated the dynamics of NF-
B translocation in living neurons by
tracing the NF-
B subunit RelA (p65) with jellyfish green fluorescent
protein. We found that green fluorescent protein-RelA was located in
either the nucleus or cytoplasm and neurites, depending on the
coexpression of the cognate inhibitor of NF-
B, I
B-
.
Stimulation with glutamate, kainate, or potassium chloride resulted in
a redistribution of NF-
B from neurites to the nucleus. This
transport depended on an intact nuclear localization signal on RelA.
Thus, in addition to its role as a transcription factor, NF-
B may be
a signal transducer, transmitting transient glutamatergic
signals from distant sites to the nucleus.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B is a
transcription factor that may act via the latter mechanism. Recently,
it was reported that potentiated synapses are marked with a molecular
tag that may sequester relevant proteins necessary for changes in gene
expression (3). NF-
B is present in synaptic compartments (6-10) and
rapidly activated independent of protein synthesis (11), making this
factor a likely candidate as a synaptic tag (3). NF-
B is present in
many neuronal cell types (for review, see Ref. 12) and, in neurons, can
be constitutively active (13-15) or activated by a variety of stimuli
such as glutamate (7, 16, 17) and amyloid
-peptide (18, 19). A
physiological role was defined for NF-
B in neuroprotection against
amyloid
-peptide (19) and oxidative stress and glutamate (20, 21). Depending on the context, NF-
B might also be involved in
neurodegeneration (22).
B DNA-binding subunits are known: p50,
p52, RelA (p65), c-Rel, and RelB (23, 24). The important role of the
transactivating RelA subunit is apparent in relA
knockout mice, for which there is a high rate of embryonic
mortality. Inhibitory subunits are I
B-
, I
B-
, I
B-
(p105), I
B-
(p100), and I
B-
(25). Within the nervous
system, heteromeric NF-
B is most frequently composed of two
DNA-binding subunits (e.g. p50 or RelA) that either are
constitutively active or form a complex with the inhibitory subunit
I
B-
(6, 7, 13, 14, 16, 18, 26). Interactive ankyrin repeats of
I
B-
can physically block the nuclear localization signal (NLS) on
the RelA subunit (27, 28), preventing transport of the complex into the
nucleus. Activation of NF-
B results in the degradation of I
B-
,
which in turn exposes the NLS, allowing NF-
B to be transported into
the nucleus (29). Thus, the specific post-translational regulation of
NF-
B and its synaptic distribution support the idea that NF-
B
functions both as a transcription factor in the nucleus, where it can
function as a molecular switch for turning on gene expression, and as
an immediate retrograde signal transducer, which unifies signal
perception at distant sites (dendrites, axons, and synapses) (6, 12,
30). It is not known, however, if NF-
B undergoes retrograde
transport upon activation.
B RelA was
transported from distant sites (neurites) to the nucleus in living cells using jellyfish green fluorescent protein (GFP) fusion technology to attach a fluorescent tag to the RelA subunit of NF-
B. To analyze the transport of NF-
B, a fusion protein was constructed that consisted of RelA and a GFP mutant optimized for maximal light emission
(EGFP). This new technique offers the opportunity to image both the
distribution and interactions of the protein in living cells. In
contrast to intracellular antibody staining, using this technique,
living cells can be observed over time, and small structures can be
labeled, e.g. the label in dendritic spines can be greatly
enhanced following overexpression of the GFP protein (31).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
B (16, 18, 35, 36).
B-
vector (38) were used. A
pcDNA3-EGFP expression plasmid was generated using
BamHI/NotI restriction of pEGFP-1 and sticky-end
ligation of the purified fragment into pcDNA3. The
pcDNA3-EGFP-RelA expression plasmid was constructed by recombinant polymerase chain reaction using Pwo/Taq DNA
polymerases (Expand High Fidelity PCR system, Roche Molecular
Biochemicals). pEGFP-1 was used as the template for polymerase chain
reaction to amplify an EGFP fragment (769 base pairs) with forward
primer A (5'-AAGCTTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTC-3') and
reverse primer B
(5'-GAAGATGAGGGGGAACAGTTCGTCGGCCCCGGCCCCCTTGTACAGCTCGTCCATGCCGAGAGTGAT-3'). At the 5'-end, the fragment carried a HindIII site and a
Kozak consensus sequence (CGCCACC) and, at the 3'-end, a sequence
encoding (Gly-Ala)2 and the first 30 base pairs from
5'-RelA. Using forward primer C (5'-GGCCGACGAACTGTTCCCCCTCATCTTCC-3')
and reverse primer D (5'-GGATCCTTAGGAGCTGATCTGAC-3'), a RelA
fragment containing the first 361 amino acids including transactivation
domain TA3, but without TA1 and TA2 (37), was amplified using a 29-base pair overlap with the 3'-end of the EGFP fragment, with the
pCMV-RelA expression plasmid used as a template. This strategy
was chosen to avoid potential toxicity via the induction of neurotoxic
NF-
B target genes. The 3'-end of the RelA fragment carried a stop
codon and a BamHI restriction site. The recombinant fragment
was ligated into the HindIII/BamHI site of the
pcDNA3 vector. The NLS of the pcDNA3-EGFP-RelA expression
vector was mutated by site-directed mutagenesis polymerase chain
reaction (QuikChange site-directed mutagenesis kit, Stratagene, La
Jolla, CA). Lys287, Lys290, and
Arg291 were mutated to threonine residues (forward primer,
5'-GTCACCGGATTGAGGAGACACGTACAACGACATATGAGACCTTCAAG-3'; and reverse
primer, 5'-CTTGAAGGTCTCATATGTCGTTGTACGTGTCTCC TCAATCCGGTGAC-3'). Mutations were verified by sequencing.
80 °C until used.
B-binding site
and the Sp1-binding site were labeled at the 5'-end with
-32P and T4 polynucleotide kinase. Binding reactions (20 µl) contained 20 µg of protein, 1 µg of poly(dI-dC), 15,000 cpm
labeled DNA, and probe buffer (20 mM HEPES, pH 7.9, 100 mM KCl, 20% glycerol, 0.25% Nonidet P-40, 0.5 mM EDTA, 2 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride). Binding reactions were
initiated by the addition of a DNA-binding mixture to the nuclear
extracts at room temperature. For supershift analysis, anti-RelA
antibody (Santa Cruz Biotechnology, Heidelberg) was incubated for 20 min at room temperature with the 32P-labeled
oligonucleotide probes. Reaction mixtures were resolved by
electrophoresis on a 4% polyacrylamide gel in 0.5× Tris borate/EDTA (1× Tris borate/EDTA = 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA, pH 7.2). Gels
were dried and exposed to x-ray film. For competition experiments,
32P-labeled oligonucleotide probes were mixed with a
10-fold excess of unlabeled competitor oligonucleotides prior to
incubation with the nuclear extracts.
-tocopherol (0.1 mM)-supplemented glia-conditioned medium after each exposure (31). Glutamate (100 or 500 µM) or kainate (50 or 100 µM) was added to
the glia-conditioned medium for 5 min. Cells were washed twice, and
movements of EGFP-RelA and EGFP-(NLSmut)RelA were observed for 2 h. Quantification of fluorescence intensities was performed as
described previously (40), but without background subtraction, using
IP-Lab-Spectrum software (Scanalytics, Fairfax, VA). In brief, GFP
fluorescence over distance was measured for untreated conditions
(I0) and after treatment (I) as
values obtained after integrating the area under the plotted
fluorescence intensities. For statistical evaluation, relative values
(I/I0 × 100) obtained from different
neurites and experiments were pooled and analyzed as the experimental
group containing EGFP-RelA versus the control group
containing EGFP-RelA with a mutated NLS, using the Wilcoxon rank sum
test. Pseudo-color images were created using NIH Image Version
1.61.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B RelA
tagged with EGFP--
To analyze the distribution of NF-
B, a GFP
fusion protein containing RelA with its nuclear localization signal was
constructed (Fig. 1A). This
construct contains only one weak transactivation domain, TA3 (37), to
avoid potential toxicity via induction of NF-
B target genes or via
squelching (41) of other signal transduction pathways. The feasibility
of using this GFP-RelA fusion protein to analyze nuclear transport was
tested in HEK 293 cells (Fig. 1B).
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Fig. 1.
Characterization of a fusion protein of EGFP
and RelA. A, a scheme of the construct showing
the spacer region inserted in the frame; B, distribution of
EGFP-RelA in HEK 293 cells. After transfection, EGFP fluorescence was
visualized using a microscope equipped with epifluorescence
illumination. Shown are EGFP-RelA in the nuclei and
translocation to the cytoplasm when I B-
was coexpressed. Unfused
EGFP was diffusely distributed in all cellular compartments and was not
regulated by I
B-
.
B-
resulted in the exclusion
of EGFP-RelA from the nucleus (Fig. 1B), consistent with the
idea that I
B-
regulates the location of EGFP-RelA in the cell. In contrast, distribution of unfused EGFP was not influenced by the coexpression of I
B-
(Fig. 1B).
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Fig. 2.
A, analysis of EGFP-RelA fusion protein
expression by Western blotting. HEK 293 cells were transiently
transfected and harvested after 24 h. Lanes 1-4 were
immunoblotted using an antibody directed against GFP (Roche Molecular
Biochemicals). Lanes 5-8 were immunoblotted using an
antibody to the RelA subunit of NF- B. Cell extracts derived from HEK
293 cells were transfected with an expression vector for EGFP-RelA
(lanes 1 and 5), for RelA (lanes 2 and
6), and for EGFP (lanes 3 and 7).
Extracts from mock-transfected cells were loaded on lanes 4 and 8. The migration positions of molecular mass markers (in
kilodaltons) are indicated on the left. The positions of specific
immunoreactive bands for EGFP-RelA and RelA are indicated by
arrowheads on the right. B, characterization of
DNA binding by electrophoretic mobility shift assay. Equal amounts of
high salt cell extracts were analyzed for activities retarding a
32P-labeled NF-
B probe using overexpressed EGFP-RelA
protein (control; lane 1), an antibody against RelA
(lane 2), or competing nonspecific Sp1 oligonucleotides
(lane 3) or NF-
B oligonucleotides (lane 4)
(10-fold molar excess). The positions of specific EGFP-RelA complexes
(arrowhead), a nonspecific band (
), and the unretarded
probe (arrow) are marked.
enhancer probe, the binding of EGFP-RelA was examined. The
probe bound strongly to EGFP-RelA (Fig. 2B, lane
1). Supershifting with the antibody to RelA verified the identity
of the EGFP-RelA complexes (Fig. 2B, lane 2).
Antibody binding to EGFP-RelA was specifically competitively inhibited
by unlabeled NF-
B oligonucleotide (Fig. 2B, lane
4), but not by the nonspecific Sp1 oligonucleotide (lane
3).
B-dependent luciferase vector (Fig. 3, A and B). The
fusion protein activated NF-
B-dependent transcription, whereas the control reporter, containing mutated NF-
B sites, was not
activated (Fig. 3A). Transcription mediated by EGFP-RelA was
inhibited when I
B-
was coexpressed. In general, however, EGFP-RelA exhibited a lower transactivation capability compared with
that of RelA (Fig. 3B) due to the fact that this fusion
protein contains only one weak transactivation domain (TA3) (37).
Deletion of strong transactivation domains was done to avoid potential toxicity via the induction of NF-
B target genes (42) or via squelching (41) of other signal transduction pathways. Taken together,
the biochemical and cell biological data indicate that the behavior of
the fusion protein was sufficiently similar to that of endogenous RelA
to warrant using this EGFP-RelA construct to examine transport in
living neurons.
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Fig. 3.
EGFP-RelA activates
NF- B-dependent transcription.
HEK 293 cells were transfected with an NF-
B-dependent
luciferase indicator vector or an indicator vector without NF-
B
sites (tk-luc) together with a Renilla luciferase internal
standard vector and additional expression vectors as indicated. Total
plasmid amounts were kept constant. Bars represent
means ± S.D. of relative light units from three independent
determinations. A, experiments characterizing EGFP-RelA;
B, control experiments with unfused RelA. CMV,
cytomegalovirus.
B may be regulated via this mechanism in both cell types.
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Fig. 4.
Nuclear localization of EGFP-RelA requires an
intact NLS. A, shown is a schematic localizing the NLS
mutations. Amino acids are given in one-letter code. B,
upper panels show the localization of EGFP-RelA with mutated
NLS (EGFP-(NLSmut)RelA) in neurons (left panel) and in HEK
293 cells (right panels); lower panels show the
localization of EGFP-RelA with intact NLS in neurons (left
panel) and in HEK 293 cells (right panel). Micrographs
were taken with a ×40 objective using epifluorescence
illumination.
B-
Regulates the Distribution of
EGFP-RelA--
To activate the reconstituted EGFP-RelA complex via
endogenous signal transduction pathways, it was necessary to avoid
overloading the system with an overexpression of I
B-
. Therefore,
it was essential to determine the least amount of I
B-
necessary
to keep the EGFP-RelA fusion protein in the cytoplasm (Fig.
5). The amount of the EGFP-RelA
expression plasmid was kept constant, and different amounts of
I
B-
expression plasmid were added. The ratios were calculated
based on the molecular masses of the expression plasmids. For both
hippocampal neurons (Fig. 5, a, c, and
e) and HEK 293 cells (b, d, and
f), increasing the concentration of I
B-
resulted in
changes in the distribution of EGFP-RelA, from primarily nuclear
(a and b), to nuclear or cytoplasmic for each
specific cell (c and d), and finally to primarily
cytoplasmic (e and f). In the cytoplasm,
EGFP-RelA was observed within puncta. Based on these data, a ratio of
1:0.2 (EGFP-RelA/I
B-
) was used in the time course experiments
described below.
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Fig. 5.
High levels of
I B-
induce
cytoplasmic localization of EGFP-RelA. a, c,
and e, expression in neurons; b, d,
and f, expression in HEK 293 cells. The amounts of EGFP-RelA
were kept constant, whereas the amount of I
B-
was gradually
increased. Cells were biolistically transfected with an expression
vector for EGFP-RelA in neurons (a) and in HEK 293 cells
(b), with an expression vector for EGFP-RelA and I
B-
(1:0.2 molar ratio) in neurons (c) and in HEK 293 cells
(d), and with an expression vector for EGFP-RelA and
I
B-
(1:0.33 molar ratio) in neurons (e) and in HEK 293 cells (f).
B was activated by glutamate in a
hippocampal neuron/glia co-culture (32, 33). Without treatment, cells
exhibited no nuclear staining, whereas following treatment with
glutamate, nuclear staining was visible (data not shown). To examine
whether treatment with the glutamate agonist kainate activated RelA in
these cultured cells, as previously observed in cerebellar granule
cells (16), we also examined the effects of kainate. A 5-min treatment
with 100 µM kainate resulted in a robust activation of
RelA after a delay of 90 min (Fig. 6).
The use of a monoclonal antibody specific for the activated form of
NF-
B allowed us to detect RelA in the neuronal nuclei induced by a
brief 5-min kainate treatment. In addition, residual amounts of
activated NF-
B could still be detected in neurites 90 min after a
kainate pulse (Fig. 6, right panels), similar to the
in vivo situation of hippocampal granule cells (10). In processes of hippocampal granule cells, activated NF-
B is present, most likely due to permanent neuronal activity in these cells in
vivo. Control cultures incubated with secondary (but not primary) antibody did not exhibit detectable staining (data not shown). Thus,
the hippocampal cultures used here are very well suited to study
transport of RelA.
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Fig. 6.
Kainate activates
NF- B in hippocampal neurons. Neuronal
cultures were left untreated (control (Con); left
panels) or incubated for 5 min with 100 µM kainate
and fixed after 90 min (right panels). Cell cultures were
analyzed by indirect immunofluorescence for an increase in RelA
immunoreactivity (upper panels). Nuclear
4,6-diamidino-2-phenylindole (DAPI) staining is shown
(lower panels). Scale bar = 50 µm.
B
redistribution from neurites to the nucleus. Therefore, we used
time-lapse microscopy.
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Fig. 7.
Depolarization activates translocation of
EGFP-RelA in hippocampal neurons. Cells were biolistically
transfected with an expression vector for EGFP-RelA and I B-
(1:0.33 molar ratio). Upper panel, cultures under control
conditions; lower panel, cells treated with 100 mM KCl for 5 min. After 90 min, cells were fixed and
analyzed with epifluorescence. Representative data from five
independent experiments are shown.
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Fig. 8.
Kinetic analysis of glutamate-mediated
translocation of EGFP-RelA. A, images of a hippocampal
neuron biolistically transfected with EGFP-RelA and I B-
(1:0.2
molar ratio). Numbers denote time-lapse from the initiation
of the stimulus (500 µM glutamate; panel a).
B, quantitative analysis of the translocation event.
Fluorescence intensities were measured along a thin line (white
line in panel a). Fluorescence intensities were plotted
at the time of the glutamate pulse 0 min (black line) and
100 min (gray line) following the pulse. The positions of
the nucleus, somatic cytoplasm, and neurite are marked. Similar results
were obtained from at least five cells recorded on different days.
C, images of a hippocampal neuron biolistically transfected
with EGFP-(NLSmut)RelA and I
B-
(1:0.2 molar ratio).
Numbers denote time-lapse from initiation of the stimulus
(500 µM glutamate) at 0 min (panel a) up to
2 h (panel c).
B (RelA). A strong difference was seen in the NLS
controls (Fig. 9, c and d). Here, stimulation with glutamate (Fig. 9d) resulted in the same image as under
unstimulated conditions (Fig. 9c); obviously, the blockade
of nuclear transport (NLS mutation, see Fig. 4A) interfered
also with the retrograde transport from neurites. Only a very weak
photobleaching is observed when the images in Fig. 9 (c and
d) are compared. To address the significance of the
transport described here, we performed a quantitative analysis (Fig.
10).
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Fig. 9.
Mutation of the NLS sequence interferes with
glutamate-mediated nuclear translocation. Similar results were
obtained from at least three cells recorded on different days.
a, pseudo-color image of a hippocampal neuron biolistically
transfected with EGFP-RelA under control conditions (see Fig.
8A). b, the same cell as in a,
100 min after stimulation with 500 µM glutamate. Note
increased nuclear GFP fluorescence and loss of fluorescence in
processes. c, control for photobleaching. Shown is a
pseudo-color image of a hippocampal neuron biolistically transfected
with EGFP-RelA containing a mutated NLS sequence that is not
transported to the nucleus (see Fig. 8C).
d, the same cell as in c, 2 h after
stimulation with 500 µM glutamate. Note that there were
no gross changes in GFP fluorescence in the nucleus and long processes.
The loss of the halo seen around the cell in c is due to a
slight focus shift.
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Fig. 10.
Neuritic fluorescence ratio
(I/I0) of hippocampal
neurons expressing EGFP-RelA or EGFP-(NLSmut)RelA. Neurons were
biolistically transfected with EGFP-RelA and I B-
(1:0.2 molar
ratio) or EGFP-(NLSmut)RelA (n = 5). Ratios were
measured for individual neurites from independent experiments. Values
for I0 and I were obtained after
integrating the areas under the plotted fluorescence intensities (as
detailed in the legend to Fig. 8B). The lines are positioned
over the neurites, which were identified by morphological criteria,
excluding the cytoplasm. A, fluorescence intensity was
measured from images taken before (I0) and
2 h after a 5-min stimulation with 500 µM glutamate
(I). Fluorescence ratios were significantly different
between neurites of EGFP-RelA- and EGFP-(NLSmut)RelA-expressing cells
(Wilcoxon rank sum test, p < 0.0068). Note the
decrease in fluorescence intensity (left bars) due to
retrograde transport in EGFP-RelA-transfected neurons, which was not
observed in the NLS controls (right bars). B,
fluorescence intensity was measured in neurites before and 2 h
after kainate stimulation (see A). Treatment with kainate
resulted in a significant difference in fluorescence intensity of
EGFP-RelA- and EGFP-(NLSmut)RelA-expressing cells (Wilcoxon rank
sum test, p < 0.00027).
B from
synaptic sites to the nucleus (Fig. 10, A and B,
left bars). In contrast, this decrease was not observed in
neurons transfected with EGFP-(NLSmut)RelA (right columns)
since no retrograde transport and no relevant photobleaching occurred.
The statistical significance of transported EGFP-RelA in comparison
with EGFP-(NLSmut)RelA was analyzed using the Wilcoxon rank sum test. A
highly significant difference between the experimental group
(EGFP-RelA) and the control group (EGFP-(NLSmut)RelA) was evident with
both stimuli glutamate (p < 0.0068) (Fig.
10A) and kainate (p < 0.00027) (Fig. 10B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and confirmed that this fusion protein (EGFP-RelA) retained its
functionality as a transcription factor. A subfragment with the
DNA-binding domain, the nuclear localization signal, and
transactivation domain TA3 (37) was used. This strategy was chosen to
avoid potential toxicity via the induction of neurotoxic NF-
B target
genes (42) or squelching (41) of other signal transduction pathways.
Reporter gene and DNA binding assays confirmed that this fusion protein
(EGFP-RelA) retained its functionality as a transcription factor.
EGFP-RelA was present in the nuclei of neurons; but after
overexpression together with the cognate inhibitor of NF-
B,
I
B-
, the distribution of the protein changed from nuclear to
neuritic (in dendrites and axons). In glutamate-stimulated hippocampal
neurons, a return of EGFP-RelA from a neuritic to a nuclear
distribution was observed. Interestingly, the redistribution of
EGFP-RelA was dependent on a functional NLS. We conclude that NF-
B
is capable of being a signal transducer, transmitting information from,
for example, active synapses to the nucleus, in addition to its well
known role of a transcription factor. Specifically, in mammalian
neurons, NF-
B may function as a retrograde messenger, transmitting
glutamatergic signals from distant sites to the nucleus.
B activity was observed in this
neuron/glia co-culture paradigm, in contrast to that previously observed in serum-free cultures (14). Previously, we found that the
constitutive NF-
B activity was repressed in the presence of glia in
the cultures used here (43). Overexpression of EGFP-RelA alone
or together with its cognate inhibitor protein I
B was chosen to
follow the distribution of both activated (without I
B) and inhibited
(with I
B) NF-
B. From a neuritic localization, which was induced
by the presence of I
B, the fusion protein migrated after stimulation
to the nucleus. In this line, activated endogenous NF-
B was
detected in neurites and the nucleus only after glutamatergic stimulation (see Fig. 6). Supporting this result of glutamatergic stimulation, we recently described activated NF-
B present in vivo in the dendrites and nuclei of hippocampal granule cells (10).
B was activated in
hippocampal neurons by glutamate, consistent with its functioning as a
signal transducer. This is in accordance with previous reports of the
activation of NF-
B in cerebellar granule cells via ionotropic glutamate receptors (7, 16). This observation could be extended to
hippocampal neurons (this study). Also, the stimulation of non-N-methyl-D-aspartic acid receptors via
kainate resulted in strong activation of NF-
B in cultivated
hippocampal neurons. Activation of NF-
B via
non-N-methyl-D-aspartic acid receptors using
kainate is known to result in a strong increase in major histocompatibility complex class I expression (16). Initially, it was
reported that inhibition of neuronal activity with tetrodotoxin results
in an up-regulation of major histocompatibility complex class I
expression as part of an immunosurveillance pathway to potentially
remove injured neurons (44, 45). Recently, a down-regulation of major
histocompatibility complex class I expression after intraoccular tetrodotoxin injection was observed in vivo (46). Although
these data may appear contradictory, the results were obtained using two different experimental paradigms and thus may reflect the complexity of the system. It is tempting to speculate that activation of NF-
B via glutamatergic stimulation may in turn regulate major histocompatibility complex class I expression, which may function as
synaptic glue (46). For example, in invertebrates, the inhibition of
NF-
B may function as an injury signal, whereas in mammals, injury
may activate NF-
B. In this line, Aplysia NF-
B is
inhibited after nerve crush, whereas traumatic spinal cord injury
activates NF-
B in rats (36). Future experiments may elucidate this issue.
B to transmit information from, for example,
active synapses to the nucleus is supported by many studies demonstrating the presence of NF-
B in synaptic regions. Synaptosomes can contain presynaptic proteins that are sealed and stabilized by the
postsynaptic density, and latent forms of NF-
B have been found in
synaptosomal preparations (6). Low salt extracts prepared from
synaptosomes contain NF-
B proteins such as p50 and RelA together
with I
B-
. Synaptophysin cofractionates with NF-
B proteins during purification. Colocalization of synaptophysin and NF-
B proteins has also been detected in rat cerebral cortex (6). NF-
B can
be activated with the detergent deoxycholate, resulting in two specific
DNA-binding complexes with different sensitivities for deoxycholate.
Supershifting and inhibition with recombinant I
B-
show a
bona fide DNA-binding complex that includes the RelA and p50
subunit. These data have been confirmed using hippocampal synaptosomal
preparations (8). In addition, a robust increase in RelA mRNA after
long-term potentiation has been reported in vivo (8).
It is possible that this is part of a feed-forward mechanism leading to
increased DNA binding to NF-
B elements during long-term
potentiation. Purkinje cell synapses also were analyzed using light
microscopy, and en passant synapses were found to contain
NF-
B (7). In addition, an electron microscopy study found NF-
B-
and I
B-
-like immunoreactivities within dendrites (10), including
dendritic spines and postsynaptic densities, of neurons in the
hippocampus and cerebral cortex (9). In Drosophila melanogaster, the NF-
B homolog Dorsal colocalizes with the
I
B homolog Cactus within the nervous system. Both proteins are
detected at high levels in postsynaptic sites of glutamatergic
neuromuscular junctions (47). Therefore, NF-
B and I
B-
and
their homologs in other species appear to be present in pre- and
postsynaptic regions.
B-
is degraded, the NF-
B
subunits p50 and RelA are transported into the nucleus due to the
presence of an NLS (38). The molecular basis for this masking of the
NLS by I
B-
is now evident from studies of their crystal structure
(28) showing that I
B-
covers an
-helix segment of RelA
containing the NLS. Using the Aplysia system, Ambron and
co-workers (53) have shown that axons contain NF-
B in both its
active DNA-binding and inactive forms, presumably complexed with
I
B-
. The inactive form may be activated in vitro via
treatment with deoxycholate, as described for synaptosomal NF-
B
(6).
B was translocated after glutamate, kainate, or KCl
stimulation in hippocampal neurons from neurites to the nucleus. In
this line, unidirectional movement of the transcription factor CREB was
observed after injection of CREB protein labeled with fluorescence dye
into dendrites of hippocampal neurons (54).
B was regulated in living hippocampal neurons and HEK 293 cells
via interactions with I
B-
. Moreover, we also found that the
localization of EGFP-RelA to neurites and varicosities was regulated by
I
B-
in these living cells, consistent with its being a remote
signal transducer. In conclusion, the results of this study support the
hypothesis that NF-
B is involved in translating short-term signals
from distant sites in neurites into long-term changes in gene
expression, which may have a key role in plasticity, development, and survival.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Prof. Dr. M. Frotscher for continuous support and helpful suggestions, Dr. Harald Neumann for helpful discussions, Prof. Dr. Carlos Dotti for providing essential protocols, Prof. Dr. Rainer Greger for providing chambers for the time-lapse experiments, and Dr. H. Stockinger for providing the anti-RelA monoclonal antibody. We also thank Titus Sparna for help with quantification and Elmar Böhm for superb technical assistance.
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Note Added in Proof |
---|
Recently, Freudenthal and Romano ((2000) Brain Res. 855, 274-287) have shown that training activated synaptosomal NF-KB, thus providing a further hint for synapse to nucleus signaling.
![]() |
FOOTNOTES |
---|
* This work was supported in part by the Volkswagen-Stiftung, the Deutsche Forschungsgemeinschaft, and the European Community.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Syngenta Crop Protection AG, Research
Biochemistry, 4002 Basel, Switzerland.
§ These authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 49-2302-669-129; Fax: 49-2032-669-220; E-mail: c.kaltschmidt@uni-wh.de.
Published, JBC Papers in Press, November 28, 2000, DOI 10.1074/jbc.M009253200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CREB, cAMP-responsive element-binding protein; NLS, nuclear localization signal; GFP, green fluorescent protein; EGFP, enhanced green fluorescent protein; HEK, human embryonic kidney.
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