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The Journal of Neuroscience, January 1, 1998, 18(1):26-35
Differential Intracellular Sorting of Immediate Early Gene mRNAs
Depends on Signals in the mRNA Sequence
Christopher S.
Wallace1,
Gregory L.
Lyford2,
Paul F.
Worley2, 3, and
Oswald
Steward1
1 Department of Neuroscience, University of Virginia,
Charlottesville, Virginia 22908, and Departments of
2 Neuroscience and 3 Neurology, Johns Hopkins
School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
This study characterizes the differential targeting of recently
synthesized immediate early gene (IEG) mRNAs to neuronal cell bodies
versus dendrites and tests the hypothesis that this targeting is based
on signals in the encoded proteins. A single electroconvulsive seizure
induces the expression of a number of IEG mRNAs in granule cells of the
dentate gyrus. Most of these IEG mRNAs remain in the cell body,
including two that are characterized in the present study (the mRNAs
for NGFI-A and COX-2). In contrast, the mRNA for Arc
moved rapidly into dendrites at an apparent rate of ~300 µm/hr.
Inhibiting protein synthesis with cycloheximide did not disrupt the
differential mRNA sorting, demonstrating that the differential
targeting of mRNAs is not dependent on translation.
Key words:
Arc; dendritic mRNA; mRNA localization; electroconvulsive seizure; mRNA transport; immediate early gene; dendrite
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INTRODUCTION |
Most of the mRNAs that neurons
express are restricted to the region of the cell body and very proximal
dendrites; however, a small subset of mRNAs is localized in dendrites
at relatively high levels (for a recent review, see Steward, 1997 ).
Such differential sorting of mRNA within the cytoplasm could, in
principle, occur in three general ways: (1) Most mRNAs could be
restricted to the cell body because of limited diffusion, whereas mRNAs
that move into dendrites could be delivered by selective transport
processes. (2) mRNAs could be restricted to the the cell body via some
selective tethering mechanism, whereas mRNAs that are not tethered
would diffuse into dendrites. (3) Both the restriction of certain mRNAs to the cell body and the delivery of other mRNAs into dendrites could
be attributable to selective targeting mechanisms.
In terms of these possibilities, any mechanisms that select mRNAs for
retention in the cell body or delivery into dendrites presumably
require some signals through which the different mRNAs are recognized
(what have been termed mRNA zip codes; see Steward and Singer, 1997).
These zip codes could be in the nascent protein that the mRNA encodes,
so that the mRNA is targeted together with its nascent peptide, or in
the nucleotide sequence of the mRNA itself.
A barrier to the analysis of mRNA targeting in neurons is that
most differentially sorted mRNAs are expressed constitutively so that
the ongoing process of mRNA sorting cannot be evaluated. In the present
study we document through nonradioactive in situ hybridization the very precise differential sorting of mRNAs encoding different immediate early genes (IEGs) a phenomenon that provides an
opportunity to explore the dynamic regulation of sorting of recently
synthesized mRNAs in neurons in vivo. After an appropriate stimulus (for example, a single electroconvulsive seizure, ECS), most
IEG mRNAs, like most constituitively expressed mRNAs, remain in the
region of the neuronal cell body. However, one IEG has been discovered
for which the transcript becomes localized throughout dendrites.
Because this gene encodes a protein that becomes associated with the
cytoskeleton, it was named Arc [activity-regulated
cytoskeleton-associated protein (Lyford et al., 1995); also identified
as arg3.1 (Link et al., 1995)].
The differential sorting of IEG mRNAs provides an opportunity to test
hypotheses about the nature of the mRNA zip codes. If either the
restriction of mRNAs to the cell body or the targeting of mRNAs to
dendrites depends on a zip code in the peptide, then proper sorting
will require protein synthesis. Thus, the present study evaluates
whether the differential sorting of Arc mRNA versus two
representatives of the many IEG mRNAs that remain in the cell body
(NGFI-A and COX-2) is disrupted by blocking translation with cycloheximide. Our results reveal that cycloheximide treatment, which
blocked expression of the IEG proteins, did not disrupt the
differential sorting of Arc mRNA versus NGFI-A and COX-2
mRNAs. These results indicate that the zip codes for sorting IEG mRNAs lie in the mRNAs themselves and not in the encoded peptides.
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MATERIALS AND METHODS |
Electroconvulsive seizures. ECSs were induced in male
Sprague Dawley rats 25-40 d of age (108-195 gm). Current was passed transcranially (40 mA for 0.5 sec) via electrodes pressed against the
scalp immediately posterior to the orbits or via ear clip electrodes.
This treatment resulted in a generalized tonic/clonic seizure. Animals
were allowed to survive 15 min (n = 4), 30 min (n = 6), 1 hr (n = 4), 2 hr
(n = 2), 4 hr (n = 6), 6 hr
(n = 2), 8-9 hr (n = 3), 12 hr
(n = 2), and 24 hr (n = 2).
Inhibition of protein synthesis. Cycloheximide (CHX; Sigma,
St. Louis, MO) was prepared as a 10 mg/ml solution in sterile saline.
Rats prepared for in situ hybridization received an
injection of CHX (20 or 50 mg/kg, i.p.). Injection controls
(n = 3) were allowed to survive 2 hr and 15 min or 4 hr
and 15 min after CHX. Rats in the CHX+ECS group were injected with CHX
15 min before ECS and were allowed to survive 1 (n = 2), 2 (n = 3), or 4 hr (n = 4). In
addition, some rats received CHX either immediately after ECS and
survived 1-2 hr (n = 2) or received CHX 5 min after ECS and were allowed to survive for 4 hr (n = 2). For
immunocytochemistry, rats received CHX (20 mg/kg) 15 min before ECS and
were allowed to survive for 2 hr (n = 2).
In situ hybridization. Animals were killed humanely
with an overdose of sodium pentobarbital (150 mg/kg) and perfused
transcardially with 4% paraformaldehyde in 0.1 M phosphate
buffer (PB), pH 7.3. Brains were removed and post-fixed in the
perfusate overnight at 4°C and cryoprotected in 30% sucrose in PB
for 24 hr. Then they were frozen either in powdered dry ice or over
liquid nitrogen vapors. In some cases, hemispheres from different
experimental groups were frozen together as hemi- or quadbrains. In
this way, sections from different brains could be processed together.
Sections were cut at 20 µm, using a cryostat, and thaw-mounted onto
poly-L-lysine-coated slides. Sections were stored at
 80°C until use.
To detect Arc, NGFI-A, COX-2, and  -tubulin mRNAs, we
synthesized cRNA probes. The Arc cRNA probe was transcribed
from a full-length clone of the rat Arc gene subcloned into
the pBluescript plasmid (Lyford et al., 1995). The probe for NGFI-A was
derived from the full-length coding region of rat NGFI-A subcloned in
pBluescript vector (Milbrandt, 1987), a gift of Dr. Jeffrey Milbrandt
(Washington University School of Medicine, St. Louis, MO). Probe for
COX-2 mRNA was synthesized from a nearly full-length clone of rat COX-2 subcloned into the pBluescript vector (Yamagata et al., 1993). Antisense cRNAs for Arc, COX-2, and NGFI-A probes were
transcribed from linearized plasmids by using T7 polymerase. The probe
for -tubulin was derived from a 1.25 kb portion of the coding region of chicken -tubulin mRNA (Cleveland et al., 1980) inserted in pGEM-2. Linearized plasmids were transcribed by the SP6 promoter to
yield antisense cRNA. Radiolabeled cRNA probes were prepared by
in vitro transcription in the presence of
35S-labeled uridine 5 -[ -thio] triphosphate (New
England Nuclear, Boston, MA), using the Stratagene (La Jolla, CA) RNA
transcription kit. Nonradioactive cRNA probes were synthesized with the
Ambion Maxiscript in vitro transcription kit in the presence
of digoxygenin-11-uridine-5-triphosphate (Boehringer Mannheim,
Indianapolis, IN).
In situ hybridization for radioactive cRNA probes was
performed as reported previously (Steward et al., 1990). For in
situ hybridization with digoxygenin-labeled cRNA probe, the
hybridization solution contained 2× SSC, 1% Denhardt's reagent, 10%
dextran sulfate, 0.5 mg/ml heparin, 0.5 mg/ml of yeast tRNA (Sigma),
0.25 mg/ml salmon sperm DNA (Sigma), and 50% deionized formamide.
Approximately 0.2-0.5 µg of cRNA probe was added to each section as
determined by dot blot, performed by using the manufacturer's
instructions. Digoxygenin-labeled probes were detected with the Genius
kit (Boehringer Mannheim).
Autoradiography. Sheet film autoradiograms were prepared by
exposing sections to -Max Hyperfilm (Amersham, Arlington Heights, IL) for 3 d. The slides were dipped in NT2B emulsion (Kodak,
Rochester, NY) and exposed for 7-10 d at 4°C and then developed via
D19 chemistry (Kodak). The sections were stained with cresyl violet,
dehydrated, and coverslipped with DPX (Fluka, Neu-Ulm, Germany).
Digital images of emulsion autoradiograms viewed at 100× under oil
were collected by a CCD camera linked to an Olympus Vanox microscope.
Grain density was analyzed by using the grain-counting function on the
MCID M4 software (Imaging Research, St. Catherine's, Ontario, Canada).
A Wratten 47 B (Kodak) filter was placed in the light path to reduce
the interference of the cresyl stain with grain detection. Counts were
made by using a 20 × 20 µm sample window superimposed over the
following regions of the ventral limb of the dentate gyrus. Three
frames were placed randomly over the granule cell layer, and six frames
each were placed over the proximal (within ~50 µm of the granule
cell bodies) and distal molecular layer (within ~50 µm of the pia).
Data were collected from three to four sections per animal. The raw
counts over the proximal and distal molecular layer were divided by the
counts over the cell bodies to generate an index of dendritic labeling normalized to labeling over somata expression.
Western blot analysis. Rats received a single ECS and were
killed humanely at varying times postseizure for analysis of the expression of the Arc protein, as previously described
(Lyford et al., 1995). Hippocampi were dissected and sonicated into
boiling SDS sample buffer. Then homogenates were subjected to Western analysis with a polyclonal antibody raised against a fusion protein that represents the terminal two-thirds of the Arc protein.
The experiments describing the specificity of this antibody have been reported previously (Lyford et al., 1995). As a control for equivalency of protein loading, blots also were probed with an antibody specific for the -subunit of calcium calmodulin-dependent Kinase II (Hendry and Kennedy, 1986).
Immunocytochemistry. Arc protein was detected by
using the same antibody as in immunoblots. Egr-1 protein (encoded by
the gene also called NGFI-A) was detected by using a commercial rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Rats
were killed humanely with an overdose of sodium pentobarbital (150 mg/kg) and decapitated. Brains were removed quickly, rinsed in ice-cold
PBS, pH 7.3, and frozen by immersion in 2-methyl-butane chilled on
powdered dry ice. In some cases, hemispheres from two conditions were
combined as a single block to control for processing. Frozen blocks
were wrapped in foil and stored at 80°C before cryosectioning.
Coronal sections (20 µm thickness) were retrieved on gelatin-coated
slides and processed immediately for immunocytochemistry.
Immunocytochemistry was performed with reagents provided in the ABC kit
(Vector Laboratories, Burlingame, CA). Sections were fixed in 4%
paraformaldehyde (10-20 min) in PBS, rinsed in PBS (three times for 10 min), and blocked in 10% normal goat serum (NGS) in blocking buffer
(0.5% Triton X-100 and 0.25%  -carrageenan in 0.1 M
phosphate buffer, pH 7.3) for 2 hr. Primary antibody was diluted in
blocking buffer with 5% NGS (Arc 1:100-1:250) and incubated overnight at 4°C. Sections were rinsed in PBS and then incubated for 2 hr at room temperature in biotinylated goat anti-rabbit IgG (1:100 in blocking buffer with 5% NGS). Sections were incubated in
2% ABC reagent in blocking buffer without serum for 30 min at room
temperature (Vector Laboratories) and rinsed in Tris-imidazole buffer.
Controls were processed as above except that the primary antibody was
omitted. The antibody was visualized with the ABC kit (Vector
Laboratories) with 1 mg/ml DAB (3-3 diaminobenzidine; Sigma) and
0.02% H202. Sections were dehydrated via
ethanol, cleared in xylene, and coverslipped under DPX.
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RESULTS |
Recently synthesized IEG mRNAs are targeted to different
subcellular domains
We evaluated the differential sorting of three IEG mRNAs that are
induced by intense neuronal activity: (1) Arc
(activity-regulated cytoskeleton-associated protein), (2) NGFI-A
(Milbrandt, 1987), and (3) inducible cyclo-oxygenase (COX-2; Yamagata
et al., 1993). Arc was chosen because its mRNA and the
encoded protein are targeted to dendrites (Lyford et al., 1995). NGFI-A
[also known as zif/268 (Christy et al., 1988), egr-1 (Sukhatme et al.,
1988), and Krox 24 (Lemaire et al., 1988)] was chosen because it is a
classic IEG (Sheng and Greenberg, 1990) that encodes a transcription
factor and is strongly induced by ECS in granule cells of the dentate gyrus (Cole et al., 1990). COX-2, like Arc, is a member of a
more recently identified class of IEGs that are rapidly induced by neuronal activity but that do not encode transcription factors. COX-2,
an enzyme involved in prostaglandin synthesis, was chosen because the
COX-2 protein also is localized to dendrites (Kaufmann et al.,
1996).
In nonstimulated control animals, the mRNAs for Arc, NGFI-A,
and COX-2 were present at low-to-moderate levels in neurons throughout the forebrain. The overall level of labeling in the dentate gyrus was
low for all three mRNAs, but in situ hybridization with
digoxygenin-labeled riboprobes revealed the presence of a few
relatively heavily labeled neurons within the stratum granulosum in the
case of both Arc and NGFI-A (Fig.
1). In the case of Arc, there
was also some labeling of the dendrites of the individual heavily
labeled neurons and diffuse labeling in the dendritic layers of the
hippocampus proper. Labeling in the dentate molecular layer was usually
distinctly lower than in the dendritic lamina of CA1.
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Figure 1.
Arc is the first inducible
dendritic mRNA. A, B, Views of the rat hippocampal
formation showing the distribution of Arc mRNA, using
nonradioactive in situ hybridization with
digoxygenin-labeled antisense riboprobe. C, Labeling of
the rat hippocampal formation, using a digoxygenin-labeled sense probe
for Arc mRNA. A, Basal expression of
Arc mRNA in the dentate gyrus overall is low, but a
small number of neurons that show a high level of expression appear as
occasional clusters. Note that basal levels of Arc mRNA are lower in the dentate molecular layer than in the dendritic lamina
of CA1. B, This pattern is altered dramatically by ECS when Arc mRNA is induced broadly in dentate granule
cells and migrates throughout the dendritic layer. C,
Hybridization using an Arc sense probe yielded only
background labeling. CA1 and CA3 represent the subfields of the hippocampus. DG, Dentate
gyrus. Arrowheads indicate the hippocampal fissure, the
distal limit of the dentate molecular layer. Scale bar, 250 µm.
D-I, Bright-field views of the dorsal limb of the
dentate gyrus comparing the localization of Arc mRNA
with NGFI-A mRNA and COX-2 mRNA after induction by a single ECS, using
digoxygenin-labeled antisense riboprobe. Expression of these mRNAs is
low in nonstimulated rats (D, Arc; F,
NGFI-A; H, COX-2), and all are elevated substantially by
a single ECS (E, Arc;
G, NGFI-A; I, COX-2). Note that
Arc mRNA was present throughout the dentate molecular
layer 1 hr after ECS, but NGFI-A and COX-2 mRNA were confined to the
stratum granulosa (SG) despite strong induction. This
suggests that the dendritic localization of Arc mRNA is
not simply the result of random diffusion but, rather, is the product
of selective transport. Arrowheads indicate the distal
limit of granule cell dendrites. Scale bar, 100 µm.
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The localization of Arc mRNA to dendrites is selective
After a single ECS there was a striking induction of the
mRNAs for all three IEGs that was most pronounced in granule
cells of the dentate gyrus (Fig. 1). As reported previously,
Arc mRNA was distributed rapidly throughout the dendrites of
the dentate granule cells. In contrast, the mRNAs for NGFI-A and COX-2
remained tightly localized in the cell body (Fig. 1E
vs G and I). The fact that the three IEG
mRNAs are sorted differentially demonstrates that the movement of
Arc into dendrites is not the result of random diffusion
resulting from sudden saturation of the mRNA sorting mechanisms of the
neurons.
Kinetics of Arc mRNA translocation to dendrites
in vivo
The kinetics of Arc mRNA transport were
determined by comparing Arc mRNA expression in the dentate
gyrus of naive animals with that at the following times after ECS via
in situ hybridization: 15 and 30 min, 1, 2, 4, 6, 8-9, and
12 hr (Fig. 2) and 24 hr (data not
shown). At 15 min, increases in Arc mRNA levels were obvious throughout the granule cell layer, indicating the onset of widespread transcriptional activation. Within 30 min, labeling extended well into
proximal dendrites and by 1 hr had reached the distal limit of the
molecular layer, a distance on average of ~300 µm. This pattern is
consistent with the synchronous delivery of Arc mRNA into
the dendrites of a large population of dentate granule cells; Arc mRNA appears first in granule cell somata and then
progresses radially to the pia along the major axis of granule cell
dendrites. Hence, this observed en masse translocation suggests that
Arc mRNA travels along individual dendrites at a rate of at
least 300 µm/hr. After labeling filled the molecular layer, it
continued to become increasingly dense until reaching peak levels at
2-4 hr.
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Figure 2.
Kinetics of Arc mRNA translocation
into granule cell dendrites. Sheet film autoradiograms show labeling of
a 35S-labeled cRNA probe for Arc mRNA in the
dentate gyrus at a series of time points after a single ECS. Widespread
expression of Arc mRNA in the somata of a large
population of dentate granule cells could be seen within 15 min of ECS.
By 30 min the labeling extended into the dentate molecule layer,
indicating transport of Arc mRNA into dendrites. Within
1 hr, labeling was elevated to the distal extent of granule cell
dendrites, a distance of up to ~300 µm. Levels of
Arc mRNA continued to increase, reaching a peak at 2-4 hr. By 6 hr the amount of Arc mRNA over the molecular
layer was in decline and fell to basal levels by 12 hr. Scale bar, 500 µm.
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Expression of Arc mRNA was decreased at 6-9 hr and returned
to basal levels by 12 hr. This decline of Arc mRNA labeling
appeared as a general fading of the distribution observed at its peak, with levels always highest over cell bodies.
Time course of protein expression in the hippocampal formation
after ECS
To evaluate whether expression of the Arc protein
occurred early enough that the encoded protein could play a role in
targeting, we evaluated the time course of protein induction in the
hippocampal formation after ECS by Western blot (Fig.
3). Increases in protein levels were
evident within 30 min after ECS, which is soon enough that the nascent
Arc protein could provide the signal necessary for dendritic
export of the mRNA. The translocation of newly synthesized Arc mRNA to distal dendrites was obvious by 2 hr after ECS,
so this interval was chosen to evaluate the possible role of
translation in Arc mRNA targeting.
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Figure 3.
Western blot analysis of the induction of the
Arc protein in the hippocampal formation by ECS. Rats
received a single electroconvulsive stimulus and were killed at varying
times postseizure. Hippocampi were dissected and sonicated into boiling
SDS sample buffer. Homogenates were subjected to Western analysis with
Arc-specific antibody. A clear increase in
Arc protein was observed by 30 min, and the highest
protein level was seen at 4 hr. Levels of Arc protein returned to the pre-seizure baseline by 24 hr. The blot was probed with
an antibody against -CaMKII to control for equivalency of protein
loading.
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IEG mRNAs are sorted differentially despite inhibition
of translation
To evaluate whether either the selective retention of newly
synthesized mRNAs in the cell body or the delivery of mRNAs to dendrites (or both) depends on translation, we evaluated the
differential sorting of IEG mRNAs after treatment with CHX, a compound
that blocks elongation of the nascent peptide chain. As illustrated in
Figure 4, treatment with CHX (20 mg/kg,
i.p.) 15 min before the seizure blocked the increases in IEG
protein levels that otherwise are seen after ECS. Using this paradigm,
we prepared a separate set of animals to examine IEG mRNA distribution
by in situ hybridization.
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Figure 4.
Inhibition of protein synthesis by
cycloheximide does not interrupt translocation of Arc
mRNA despite blockade of Arc protein expression.
A, B, Expression of the Arc protein shown
by immunocytochemistry in dark field 2 hr after ECS without
cycloheximide (A) and with cycloheximide
(B). Scale bar, 500 µm. C, D,
Dark-field views of autoradiograms comparing Arc mRNA
expression 2 hr after ECS (C) and the same
treatment 15 min after cycloheximide injection
(D). Scale bar, 500 µm. E,
Comparison of the extent of Arc mRNA translocation in
the dentate molecular layer in 2 hr ECS and 2 hr CHX+ECS animals. Grain
counts of Arc mRNA labeling over the proximal and distal molecular layer were normalized to the counts present over the granule
cell layer. Error bars show mean ± SD.
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Two hours after animals received an injection of CHX alone (20 or 50 mg/kg, i.p.) the pattern of Arc mRNA expression within the
dentate gyrus was comparable to that of controls, with labeling limited
to occasional sets of neurons. Expression of Arc mRNA did
appear somewhat elevated in neocortex and the CA1 field, however (data
not shown). Under protein synthesis inhibition by CHX, Arc mRNA was still induced by ECS in the dentate gyrus and exhibited a
clear dendritic translocation similar to that observed in rats receiving ECS alone (compare Fig. 4C and D).
Interestingly, levels of Arc mRNA expressed by dentate
granule cells were ~30% lower in CHX-pretreated animals 2 hr after
ECS (n = 2) than after ECS alone (n = 2). For this reason, levels of Arc mRNA present in the
proximal and distal molecular layer were normalized to the level
present over granule cells by using automated grain counting (Fig.
4E). If targeting of Arc mRNA into
dendrites was blocked by inhibiting protein synthesis, the relative
amount of labeling over dendrites should be decreased substantially.
However, the ratio of mRNA in dendrites to that in cell bodies was
quite similar in CHX-treated and control animals; thus, CHX treatment
had no detectable effect on dendritic translocation per se.
Inhibition of protein synthesis also did not disrupt the selective
retention of the mRNAs for NGFI-A and COX-2 in neuronal cell bodies
(see Fig. 5 for NGFI-A). Moreover, there
was no detectable change in the subcellular localization of a
noninducible mRNA (-tubulin mRNA) at 2 or 4 hr after CHX+ECS. These
results are important in the light of previous findings that prolonged
exposure of cultured hippocampal neurons to protein synthesis
inhibitors (6-12 hr) resulted in the drift of mRNAs normally
restricted to the cell body into the dendritic compartment (Kleiman et
al., 1993). These findings raised the possibility that mRNAs that were not assembled into a translational complex might diffuse nonselectively into dendrites. The failure to see any such delocalization in the
present experiments indicates that the mechanisms that underlie the
differential sorting of mRNAs in neurons in vivo remain
fully operational even when protein synthesis is blocked.
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Figure 5.
Newly synthesized NGFI-A and Arc
mRNA are sorted to their appropriate compartment after inhibition of
protein synthesis. A-D, Sheet film autoradiograms of a
hemibrain composed of the left hemisphere of an animal 2 hr post-ECS
and the right hemisphere of a 2 hr post-ECS animal pretreated with
cycloheximide (20 mg/kg) 15 min before ECS. Expression of NGFI-A mRNA
was superinduced by ECS in the presence of cycloheximide
(B) relative to ECS alone (A) but remained restricted to the granule cell
layer. Arc mRNA showed the normal pattern of dendritic
translocation (C) despite pretreatment with
cycloheximide (D). Scale bar, 2.5 mm.
Insets, Higher magnification of the hippocampal
formation from A-D. Scale bar, 500 µm. E,
F, Nonradioactive in situ hybridization of
adjacent sections to compare the cellular distribution of NGFI-A
(E) and Arc (F) mRNA
2 hr after ECS in an animal pretreated with cycloheximide. The
maintained localization of NGFI-A mRNA over dentate granule cell bodies
indicates that mRNA does not diffuse freely in dendrites when it is not
part of a translational complex. The migration of Arc
mRNA throughout the dendritic lamina therefore is consistent with its
selective transport. SG, Stratum granulosa. Scale bar, 50 µm.
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After CHX, levels of Arc mRNA are induced less strongly and decline
more rapidly
Superinduction of mRNA during translation inhibition is a feature
characteristic of IEGs in general (Sheng and Greenberg, 1990) and in
fact one that was exploited to identify Arc by differential cloning (Link et al., 1995; Lyford et al., 1995). It is noteworthy, however, that Arc mRNA was less strongly induced in dentate
granule cells after CHX+ECS despite the fact that it appears to be
superinduced in other parts of the hippocampus and in the cortex (Fig.
5). Although clearly induced by ECS in the presence of CHX, levels of
Arc mRNA in dentate gyrus neurons were actually somewhat
lower in CHX/ECS animals than in ECS animals. The finding that full expression of Arc mRNA requires protein synthesis suggests
that Arc behaves as both an IEG and a secondary response
gene in dentate granule cells. This is similar to other mRNAs induced
by ECS (Lauterborn et al., 1996). These differences were relatively
subtle during the initial period of translocation but were clear by 4 hr: ECS rats showed peak induction, whereas CHX+ECS showed marked
declines in Arc mRNA levels (Fig.
6B).
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Figure 6.
In situ hybridization for NGFI-A,
Arc, and -tubulin mRNA in 4 hr ECS and 4 hr CHX+ECS
animals. A-C, Horizontal sections of hemibrains
composed of the left hemisphere of a 4 hr ECS animal and the right
hemisphere of a 4 hr CHX+ECS animal. Neighboring sections compare the
distribution of NGFI-A mRNA (A),
Arc mRNA (B), and -tubulin mRNA
(C), using 35S-labeled antisense
riboprobes. Scale bar, 0.5 cm. D-G, Expression of
NGFI-A mRNA. H-K, Arc mRNA in the
dentate gyrus shown by in situ hybridization, using
digoxygenin-labeled antisense riboprobes. These views of the
hippocampal formation are taken from neighboring sections of the same
"quadbrain" composed of hemispheres from four experimental
conditions mounted as a single block and sectioned and hybridized
together. Shown are basal expression (D,
H), 4 hr after ECS (E,
I), cycloheximide (20 mg/kg) 15 min before ECS (F, J), and cycloheximide
injection (20 mg/kg) 5 min after ECS (G,
K). Scale bar, 750 µm.
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In contrast to what was seen with Arc, CHX did not diminish
the seizure-induced response of NGFI-A in the dentate gyrus. Indeed, inhibition of protein synthesis by CHX, whether administered before ECS
(Fig. 6F) or introduced after ECS (Fig.
6G), resulted in prolonged elevation of NGFI-A mRNA but a
decrease in Arc mRNA (Fig.
6J,K). The fact that
Arc mRNA decreased even when CHX was given after ECS
indicates that CHX does not exert its effect by reducing seizure intensity.
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DISCUSSION |
The purpose of the present study was to determine whether newly
synthesized IEG mRNAs were sorted to cell bodies versus dendrites on
the basis of signals in their mRNAs or in the nascent proteins. By
massively upregulating IEG mRNAs, a single ECS induced mRNAs localized
to both cell bodies (NGFI-A, COX-2) and to dendrites (Arc).
These mRNAs were still sorted appropriately when protein synthesis was
blocked during the period of induction and targeting, indicating that
the targeting of these mRNAs to different subcellular domains cannot
depend on a signal or signals within the nascent peptides. Instead,
these results point to signals within mRNA itself for both the
retention of newly synthesized mRNAs within cell bodies and the
targeting to dendrites.
Approaching this question in vivo and using IEG mRNA offer a
number of unique advantages over other possible strategies. In the
first place, the assessment of IEG mRNAs allows an evaluation of the
differential sorting of newly synthesized mRNAs and an analysis of the directionality and kinetics of movement of the mRNAs
from their site of synthesis. The examination of the kinetics of
movement is not possible with any constituitively expressed mRNA,
including mRNA constructs introduced into neurons in vitro by using transfection techniques. Even the observation of the movement
of RNA transcripts that have been introduced by microinjection (Muslimov et al., 1997; Prakash et al., 1997) has the potential disadvantage that the introduced mRNAs do not engage the sorting machinery where they normally would (at the nuclear pore just after
leaving the nucleus). Another advantage is that the assessment involves
native mRNAs rather than genetically modified transcripts; this
eliminates any concern about whether the sorting and transport machinery can deal appropriately with an abnormal mRNA or whether the
levels of expression of genetically manipulated transcripts may
overwhelm the sorting or transport machinery because of the use of
unusual promoters. Finally, studies in vivo have revealed a
range of mRNA localization patterns (for example, a differential subcellular distribution of the mRNAs for MAP2 and -CaMKII kinase; Steward and Wallace, 1995). So far, such precise distribution patterns
have not been seen in the in vitro setting, raising the concern that localization mechanisms in neurons in vitro may
not be fully developed.
All of this is not to discount the advantages of other approaches. In
particular, identification of the necessary and sufficient mRNA
sequence that represents a particular "zip code" will require the
introduction of candidate sequences along with some marker. Consequently, the present approach should be viewed as complementary to
others, not a substitute.
There are, however, some technical issues to consider in terms of the
in vivo approach. First, the logic of the experiment requires an effective block of the synthesis of the IEG proteins. In
this regard, the dose of CHX used was higher than what has been shown
previously to block the synthesis of IEG proteins (Hughes et al.,
1993). Because CHX inhibits peptide elongation, rather than initiation,
it remains a formal possibility that very short polypeptide chains
could be formed in the presence of CHX. In this case, however, one
still would expect a substantial disruption of mRNA sorting, manifested
by either a diminution of the transport of Arc into
dendrites or a drift of NGFI-A and COX-2 mRNAs into dendrites. Neither
effect was seen, suggesting that the zip codes that determine the
localization of IEG mRNAs reside in the nucleotide sequence and not in
the nascent protein.
The conclusion that IEG mRNAs are sorted on the basis of signals in the
mRNA itself is consistent with a growing body of evidence in other
systems. For example, all of the mRNAs that are localized differentially in oocytes and developing embryos apparently are targeted on the basis of signals in the mRNAs themselves, often in the
3-untranslated region (UTR) (for review, see Wilhelm and Vale, 1993;
St. Johnston, 1995). The limited evidence that exists regarding the
signals that target other neuronal mRNAs is also consistent with
targeting signals in the mRNAs themselves. For example, transgenes
consisting of L7 mRNA without translation initiation sites are still
properly localized in dendrites of cerebellar Purkinje cells in
vivo despite the absence of the nascent L7 peptide (Bian et al.,
1996). Similarly, transgenes made up of the 3-UTR of the -subunit
of calcium calmodulin-dependent Kinase II (-CaMKII kinase) and
bacterial -galactosidase are properly targeted to dendrites in
transgenic mice (Mayford et al., 1996). Vasopressin mRNA also appears
to be targeted into dendrites on the basis of signals in the mRNA.
Constructs containing labeled fragments of vasopressin mRNA injected
into sympathetic neurons are properly sorted into dendrites, although
the transcripts are not configured for translation of the cognate
peptide (Prakash et al., 1997). It is important to note, however, that
there may be exceptions to the rule. It remains possible, for example,
that the mRNA for MAP2 is targeted to dendrites on the basis of a
signal or signals in the nascent peptide (Marsden et al., 1996).
In many cases, signals necessary and sufficient for mRNA localization
are present in the 3-UTR portion of the mRNA. It is thus noteworthy
that there are no regions of obvious homology among the 3-UTRs of
Arc, L7, and -CaMKII kinase (three mRNAs that are
targeted to dendrites), suggesting that localization in dendrites is
not determined by a single consensus sequence. Given that the 3-UTRs
of some localized mRNAs contain separate sequences that operate at
distinct steps in targeting (for review, see St. Johnston, 1995), the
ultimate positioning of mRNAs within dendrites may involve multiple
levels of signals and associated factors.
Rate of Arc mRNA transport along dendrites
We interpret the rapid translocation of Arc mRNA into
the dendritic laminae of the dentate gyrus as representing an active and selective dendritic transport. The alternative possibility, that
Arc mRNA diffuses into dendrites after transcription whereas all other mRNAs are somehow bound within the soma, seems much less
likely. In the first place, experimentally induced seizures massively
induce the expression of a host of mRNAs. ECS induces a set of IEGs
numbering at least 20 (Lanahan et al., 1997), and at least 52 genes are
induced by kainic acid administration (Nedivi et al., 1993). Yet, to
our knowledge, all of these mRNAs that have been characterized by
in situ hybridization are localized to cell bodies except
Arc. It is hard to imagine a selection mechanism that could
restrict this heterogeneous population of mRNAs to the cell body and
yet somehow spare Arc. This is especially true because
association with the translational machinery cannot account for the
restriction, because NGFI-A and COX-2 mRNAs remain confined to the cell
body despite CHX treatment before ECS. Moreover, there is other
evidence that the mRNAs present in the dendrites of neurons in culture
are associated with microtubules (Bassell et al., 1994). This is
consistent with data in other systems implicating microtubules in the
delivery of mRNAs to particular subcellular domains (Macdonald and
Struhl, 1988; Mowry and Melton, 1992; Kislauskis et al., 1993, 1994;
Wilhelm and Vale, 1993; St. Johnston, 1995).
The translocation of Arc mRNA from granule cell bodies to
dendrites after ECS provided the first opportunity to measure the rate
at which particular mRNAs migrate in dendrites in vivo. The rate seen (~300 µm/hr) is ~10 times faster than the rate of
migration previously estimated from pulse-chase studies, using
tritiated uridine to label newly synthesized RNA (Davis et al., 1987,
1990). Unlike in situ hybridization for a specific mRNA,
however, incorporation labels a heterogeneous pool of RNAs predominated
by nonmessenger nonmessenger RNAs, with ribosomal RNA accounting for
~80% of label (Darnell et al., 1986).
The rate we observed is approximately comparable to that reported for
the movement of labeled mRNA granules in the dendrites of neurons in
culture (360 µm/hr; Knowles et al., 1996) and to the rate of movement
of radiolabeled BC1 RNA after microinjection into cultured sympathetic
neurons (~250 µm/hr; Muslimov et al., 1997). Studies of the
movement of labeled myelin basic protein mRNA after microinjection into
oligodendrocytes indicate a somewhat slower rate of 72 µm/hr (Ainger
et al., 1993), however. Our estimates are based on time sampling of the
apparent rate of movement from a lamina containing cell bodies into a
neuropil region containing a dense network of dendrites, whereas the
other studies directly measure the movement of individual packets of
mRNA in single processes. Taking this into account, the similarity in
rates is more striking than the differences.
The present findings extend our understanding of the mechanisms that
lead to a differential localization of mRNAs in vivo and
provide new clues about the nature of the transport mechanisms that
deliver mRNAs into dendrites. Although it remains to be determined if
the trafficking mechanisms that operate for IEG mRNAs are the same or
different from the mechanisms that operate for other differentially sorted mRNAs, analysis of the sorting of IEGs provides a currently unique level of access to the dynamic process of mRNA localization.
| |
FOOTNOTES |
Received Sept. 4, 1997; revised Oct. 7, 1997; accepted Oct. 9, 1997.
This work was supported by National Science Foundation Grant
IBN92-22120 and National Institutes of Health Grant N512333 (O.S.), National Institutes of Health Grant MH 53603 (P.W.), and National Institutes of Health/National Institute of Neurological Diseases and
Stroke Individual National Research Service Award NS0973 (C.S.W.). We
thank Leanna Whitmore, Angela Vames, Josh Ajima, and Douglas J. Davis
for technical assistance; Ginger Withers for discussion of this
manuscript; and Michele Paradies for providing protocols and assistance
with in situ hybridization with digoxygenin-labeled cRNA
probes.
Correspondence should be addressed to Dr. Oswald Steward, Department of
Neuroscience, University of Virginia School of Medicine, Box 5148 MR4,
Charlottesville, VA 22908.
| |
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Top
Abstract
Introduction
