BIOLOGY OF REPRODUCTION 70, 277–281 (2004)
Published online before print 1 October 2003.
DOI 10.1095/biolreprod.103.022491
M i n i r ev i ew
Activity-Dependent Regulation of Neurohormone Synthesis and Its Impact on
Reproductive Behavior in Aplysia1
Nancy L. Wayne,2,3 Wenjau Lee,3 Stephan Michel,3 John Dyer,4 and Wayne S. Sossin4
Department of Physiology,3 David Geffen School of Medicine at UCLA, Los Angeles, California 90095-1751
Department of Neurology and Neurosurgery,4 Montreal Neurological Institute, McGill University, Montreal, Quebec,
Canada H3A 2B4
ABSTRACT
uations of intense demand for release involve a requirement
for rapid stimulation of synthetic pathways to replenish releasable stores of that peptide for sufficient secretion. Secretion of peptides from neurosecretory or excitable endocrine cells is regulated by membrane excitability (i.e., membrane depolarization and/or action-potential firing) [1].
Likewise, membrane-depolarizing treatments have been
shown to stimulate the synthesis of peptides/proteins, primarily through upregulating gene transcription [2]. These
findings have generated interest in understanding possible
mechanistic links between membrane excitability, peptide
synthesis, and peptide secretion.
The reproductive neuroendocrine cells, or bag cell neurons (BCNs), of the marine mollusk Aplysia californica are
an excellent model for studying the cellular and molecular
bases of reproductive behavior, neurohormone synthesis,
and neurohormone secretion because of their large size and
accessibility [3, 4]. Furthermore, considerable information
is available regarding the predominant hormone, egg-laying
hormone (ELH), that is synthesized and secreted by the
BCNs, including the family of genes that express the preprohormone of ELH and its proteolytic processing to final
peptide products [5, 6].
After a general review of the Aplysia BCN model system, we will consider the fate of newly synthesized ELH
and its role in physiology and behavior as well as the effects of membrane excitability on transcription of the ELH
gene and translation of ELH mRNA. Finally, we will discuss a novel translational mechanism for selective ELH
synthesis that controls reproduction.
The bag cell neurons (BCNs) of the mollusk Aplysia californica provide a simple model system for investigating cellular and
molecular events regulating synthesis and secretion of a reproductive neuropeptide and their impact on physiology and behavior. The BCNs secrete a large amount of egg-laying hormone
(ELH) in response to an electrical afterdischarge. The afterdischarge also triggers cellular and molecular events leading to
upregulation of ELH biosynthesis to replenish the supply of releasable hormone that was lost because of secretion. In the present review, we discuss signal-transduction events that link
membrane excitability to ELH biosynthesis. We present evidence
that the afterdischarge stimulates ELH synthesis by upregulating
translation of ELH mRNA rather than by activating ELH gene
transcription. This increase in ELH synthesis is accompanied by
a decrease in total protein synthesis, suggesting that the synthetic machinery is being funneled selectively toward ELH. We
also discuss work showing that afterdischarge-induced ELH synthesis uses a novel mechanism of translation initiation, one involving a switch from cap-dependent to cap-independent translation initiation that activates an internal ribosome entry site
(IRES) located in the 59-untranslated region of ELH mRNA. The
IRES-regulated translation provides a unique cellular mechanism
to selectively upregulate synthesis of a critical reproductive hormone at the expense of nonessential proteins.
behavior, neuroendocrinology, neuropeptides, signal transduction
INTRODUCTION
Peptides and proteins destined for secretion are stored in
secretory vesicles; therefore, a temporal dissociation can
occur between their synthesis and release. Nevertheless, sit-
BCN MODEL SYSTEM
Series of Events Leading to ELH Secretion
Supported by grants from the National Institutes of Health (grants NS
33548 and HD 28336) to N.L.W., the Whitehall Foundation (grant F9835) to N.L.W., the Physiology Department at UCLA to N.L.W., and the
Canadian Institute of Health Research (grant MOP-15121) to W.S.S.
2
Correspondence: Nancy L. Wayne, Department of Physiology, 53-231
CHS, David Geffen School of Medicine at UCLA, 10833 Le Conte Ave.,
Los Angeles, CA, 90095-1751. FAX: 310-206-5661;
e-mail: [email protected]
1
As shown in Figure 1, the bilateral BCN clusters are
located just rostral to the abdominal ganglion of the Aplysia
central nervous system. The BCNs possess some unusual
characteristics that make them a valuable model system for
investigating their physiological properties. Each cluster
contains approximately 400 electrotonically coupled BCNs
and no other neuronal cell type [7, 8]; therefore, the researcher has access to a pure population of neuroendocrine
cells. Because of this coupling, the electrical activity of a
single BCN represents that of the entire population of neurons within a cluster. The BCNs, like most other molluskan
neurons, are very large in size, with mature BCNs measur-
Received: 20 August 2003.
First decision: 10 September 2003.
Accepted: 24 September 2003.
Q 2004 by the Society for the Study of Reproduction, Inc.
ISSN: 0006-3363. http://www.biolreprod.org
277
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WAYNE ET AL.
At a number of points, ELH synthesis can be influenced
by the afterdischarge. Membrane excitation could stimulate
transcription of the ELH gene, translation of ELH mRNA,
and/or processing of the prohormone to its final peptide
products. Earlier work by Azhderian and Kaczmarek [24]
showed evidence for the latter event but did not rule out
the first two possibilities. The remainder of the present review will describe work investigating the fate of newly synthesized ELH and the effects of afterdischarge on ELH
gene transcription and ELH mRNA translation.
FIG. 1. Sequence of events leading to ELH secretion and egg-laying behaviors. Details are given in the text. Representative sampling from an
electrical afterdischarge was recorded using a sharp microelectrode and
standard intracellular electrophysiological techniques. CNS, Central nervous system.
ing as much as 100 mm in diameter. This large size is advantageous for both electrophysiological recording and microinjection of chemicals into the neurons without damaging them. Typically, the BCNs are electrically silent, but in
response to brief electrical stimulation (seconds), they will
show a pattern of repetitive action-potential firing (10–30
min) called an electrical afterdischarge [9]. In the intact
animal, electrical signals from higher-order neurons in the
head ganglia are transmitted along the pleurovisceral connective nerves to the BCNs, eliciting an afterdischarge [10].
This afterdischarge triggers release of 1–2 mg of ELH over
the course of 1–2 h [11]. The ELH then acts at target sites
in the ovotestis to stimulate ovulation [12, 13] and in the
central nervous system to alter behaviors associated with
egg laying [14, 15]. Injection of either purified or synthetic
ELH can stimulate egg-laying behavior that is indistinguishable from the endogenously triggered response [16,
17], indicating a single peptide can activate a series of
events that culminate in a complex and prolonged behavioral repertoire. Given that Aplysia lay eggs up to once a
day during the breeding season in response to ELH [18],
the synthetic machinery in the BCNs must be upregulated
to replenish the releasable supplies of hormone for the next
egg-laying event. This will be discussed in more detail later.
ELH Gene and Peptide Processing
Earlier work showed that the bag cell peptides, including
ELH, are encoded by a family of genes [6]. The 36-aminoacid ELH peptide is derived from a larger, 271-amino-acid
preprohormone [5]. The first 29 amino acids of the preprohormone contain a signal sequence. The 242-amino-acid
prohormone is cleaved at eight internal sites to generate
nine peptide products—several of which have been shown
to possess biological activity [19, 20]. The ELH and bag
cell peptides derived from the prohormone are differentially
packaged into dense core vesicles and are transported to
different neural processes [21–23].
FATE OF NEWLY SYNTHESIZED ELH
Aplysia californica live for only 1 yr, and during the
breeding season, they will lay eggs up to once a day (in
response to afterdischarge-induced ELH secretion) for a
few weeks until the animal dies [18, 25]. Under these conditions of high demand, the BCNs lose 1–2 mg of ELH
each day because of secretion. The synthetic machinery of
these neurons has to be greatly stimulated to replenish the
supply of releasable hormone so that animals can fulfill
their reproductive agenda. Earlier work described seasonal
changes in the rate of ELH synthesis, with maximal synthesis occurring during the breeding season [26]. This finding suggests that ELH synthesis is upregulated during a
time of maximal reproductive activity. Later studies investigated the pattern of release of newly synthesized ELH and
total ELH (older 1 new) in response to stimulation of daily
afterdischarges to determine the fate of newly synthesized
ELH and its contribution to the total pattern of ELH secretion [27]. The results showed that newly synthesized ELH
is preferentially secreted within 24 h of being made [27].
This phenomenon of ‘‘last-in, first-out’’ has been observed
in a wide array of cell types, including pancreatic beta cells
[28], parathyroid cells [29, 30], pituitary cells [31, 32], and
sympathetic ganglia [33]. It is not clear how a cell can
distinguish different ages of secretory granules. However,
it is known that the BCNs discriminate between different
classes of secretory granules containing different bag cell
peptide products cleaved from the same precursor peptide
and transport them to different cellular locations [22, 23].
It has been suggested that secretory granules containing
new and older ELH might also be differentially transported
under similar types of sorting mechanisms, thus leading to
differences in the release of new versus older hormone [27].
Preferential release of newly synthesized peptide is only
biologically significant if it contributes to a large proportion
of the total amount of peptide secreted and if consequences
result from inhibiting the secretion of newly synthesized
peptide. Blocking protein synthesis in BCNs results in a
50% decrease in total ELH secreted, suggesting that newly
synthesized ELH contributes to half the total ELH secreted
[27]. This is consistent with findings in another endocrine
tissue, the parathyroid gland, in which newly synthesized
parathyroid hormone makes up 50% of the total amount of
hormone secreted [34]. Furthermore, without the contribution of newly synthesized ELH, stimulating secretion of
older ELH only is insufficient to trigger ovulation and egglaying behaviors [27]. To our knowledge, no evidence exists for changes in the structure or function of ELH as the
hormone gets older; based on analysis of the effects of different doses of ELH on egg-laying behavior [35], the most
straightforward explanation for this finding is that the
amount of older ELH that is secreted in response to afterdischarge is insufficient to stimulate egg laying. The outcome from these experiments indicates that newly synthesized ELH plays a critical role in maintaining sufficient
CONTROL OF NEUROHORMONE SYNTHESIS
279
supplies of releasable hormone for activating reproductive
physiological and behavioral events.
ACTIVITY-DEPENDENT ELH SYNTHESIS:
TRANSCRIPTION- OR TRANSLATION-MEDIATED
EVENT?
Given the biological significance of newly synthesized
ELH, it is important to understand the mechanisms by
which ELH synthesis is regulated so that supplies of releasable hormone are maintained during times of high demand. A series of experiments were conducted to test the
hypothesis that the afterdischarge, which triggers loss of
ELH through secretion, ultimately replenishes releasable
pools of hormone by stimulating ELH biosynthesis [36]. It
was shown that within 4 h after onset of the afterdischarge,
a 2-fold increase occurred in ELH synthesis compared to
that in unstimulated control tissue. This increase was seen
at the level of the prohormone, intermediate processing
products, and final ELH peptide. The stimulus-induced increase in ELH biosynthesis persisted for at least 8 h and
then declined to baseline levels by 12 h. This finding is
consistent with earlier work showing that treating BCNs
with a depolarizing high-K1 medium increased ELH synthesis [37]. Using Northern blot analysis, it was found that
the afterdischarge had no effect on the level of ELH mRNA
for up to 8 h after stimulation; therefore, afterdischargeinduced stimulation of ELH synthesis was not caused by
enhanced transcription of the ELH gene [36]. Rather, the
stimulatory effect of afterdischarge on ELH synthesis occurs at the level of translation of an abundant pool of ELH
mRNA. This makes sense given that the half-life of ELH
mRNA is greater than 32 h [36]; therefore, it would be
more effective to regulate rapid ELH synthesis through
translation of already existing mRNA rather than through
transcription of the ELH gene. Most studies investigating
regulation of neuropeptide biosynthesis have focused on
regulation of gene transcription [2]. However, examples can
be found of translation playing an important role in regulating glucose-induced synthesis of insulin prohormone in
pancreatic beta cells [38], electrical stimulation of myosin
heavy-chain synthesis in cardiocytes [39], and serotonininduced synaptic facilitation in Aplysia sensorimotor neuron connections [40, 41].
Importantly, transcription of some unidentified, nonELH gene appears to play an important role in mediating
the effect of afterdischarge on ELH biosynthesis. Treatment
of BCNs with a transcription inhibitor had no effect on the
level of ELH mRNA, had a significant inhibitory effect on
basal ELH synthesis, and completely blocked the afterdischarge-induced increase in ELH biosynthesis [36]. Transcription of a non-ELH gene likely is required for afterdischarge-induced upregulation of translation of ELH mRNA.
A ROLE FOR CAP-INDEPENDENT, INTERNAL
RIBOSOME ENTRY SITE-MEDIATED TRANSLATION
INITIATION
Cap-dependent initiation is the major translation initiation pathway in eukaryotes. As a result, it was assumed
that the afterdischarge would stimulate a conventional
translation initiation cascade to upregulate ELH synthesis
and that, in the process, total protein synthesis in the cell
would also be stimulated. However, that was not the case.
Four hours after onset of the afterdischarge, when ELH
synthesis was stimulated, a significant decline was observed
in total protein synthesis [36]. Because total protein syn-
FIG. 2. Strategy for determining if the ELH 59-UTR contains IRES activity.
Details are given in the text. A, Top) Schematic diagram of the plasmid
encoding mRNA. Shaded box indicates eBFP sequence, and hatched box
indicates enhanced green fluorescent protein (eGFP) sequence. The ELH
59-UTR is inserted between. A, Bottom) Expected outcomes depending
on whether the 59-UTR contains IRES activity. Shaded circle represents
blue fluorescence, and hatched circle represents green fluorescence.
‘‘None’’ refers to no fluorescence above background. B) Representative
pair of BCNs in which one neuron was injected with bicistronic construct
containing 59-UTR in the reverse orientation (showing no IRES activity);
the other neuron was injected with the bicistronic construct containing
59-UTR in the forward orientation (showing IRES activity). Magnification
340.
thesis was inhibited by the afterdischarge, this suggests that
important components of the cap-dependent initiation cascade were also inhibited. In cap-dependent initiation, the
eukaryotic initiation factor (eIF) 4E binds to the 59-terminal
cap of the mRNA and, via its interaction with eIF4G, positions the 40S ribosome subunit at the 59-end of the
mRNA. This allows the 40S subunit to scan the entire 59untranslated region (UTR) and locate the first appropriate
AUG start codon, at which point the 60S ribosome subunit
joins the 40S subunit to form an 80S ribosome competent
for translation elongation—and for making of a protein or
peptide. The phosphorylation state of eIF4E is thought to
be important for activating this entire process of translation
initiation [42].
Given that the afterdischarge inhibits total protein synthesis and that phosphorylation of eIF4E is a key event in
translation initiation, it was hypothesized that the afterdischarge leads to dephosphorylation of eIF4E. Using antibodies specifically directed against the eIF4E phosphorylation site in either the phosphorylated or unphosphorylated
states, Western blot analysis revealed that afterdischarge
caused a significant decrease in levels of phosphorylated
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WAYNE ET AL.
eIF4E, with no significant change in levels of unphosphorylated eIF4E [43]. This dephosphorylation of eIF4E could
explain the inhibitory effect of afterdischarge on total protein synthesis. However, how is translation of ELH mRNA
specifically stimulated at a time when both conventional
translation initiation activities and total protein synthesis
are inhibited? Obviously, something unconventional is going on here.
Evidence for what might be allowing the BCNs to funnel
their synthetic machinery toward ELH production and away
from nonessential proteins comes from the viral literature.
Viruses commonly use a cap-independent, internal ribosome entry site (IRES) to control protein synthesis; this
mechanism does not require the same complement of conventional initiation factors as does cap-dependent translation [44]. On infection of a host cell, viruses can block the
ability of the eIF4F complex to bind to the 40S subunit,
thereby inhibiting cap-dependent translation initiation of
the cell’s own proteins. On the other hand, viral mRNAs
contain IRES in the 59-UTR, which allows the ribosomes
that are now more freely available because of inhibition of
cellular cap-dependent translation to bind to the viral IRES
using a cap-independent mechanism [44]. During the past
few years, several reports have described IRES in the 59UTR of cellular mRNAs—some of which show enhanced
activity during cellular stress or when cap-dependent translation is inhibited [44–47].
The hypothesis that was tested with BCNs is that the
afterdischarge activates a cap-independent, IRES-mediated
mechanism in the 59-UTR of ELH mRNA, thereby stimulating ELH synthesis [43]. Because IRES activity in 59UTR is dependent on its three-dimensional folding rather
than on a particular base sequence, the most effective strategy to identify IRES activity has been the use of bicistronic
RNAs with two open reading frames that do not overlap
and the 59-UTR containing a putative IRES sandwiched
between [44]. The 59-UTR of ELH is long (311 base pairs)
and has a complex secondary structure, making it a good
candidate for containing an IRES. A bicistronic construct
was made containing enhanced blue fluorescent protein
(eBFP), the 59-UTR of ELH mRNA, and enhanced green
fluorescent protein in tandem and was inserted into the
pNEX vector (Fig. 2). Additional constructs were made,
including the 59-UTR in the reverse orientation and no 59UTR as a test for specificity of the response. The eBFP
reflects cap-dependent activity and is constitutively active,
also serving as a loading control. The rationale is that if
only cap-dependent translation initiation occurs and no
IRES is present, then there should be only blue fluorescence
and not green fluorescence—because nothing in the 59-UTR
insert would otherwise allow translation initiation to take
place. However, if both blue and green fluorescence occur,
then this provides evidence of an IRES in the 59-UTR (Fig.
2). All BCNs that were injected with the bicistronic construct with the 59-UTR in the reverse orientation showed
significant blue fluorescence above background but no significant green fluorescence; therefore, the 59-UTR in the
reverse orientation did not contain IRES activity. On the
other hand, all neurons that were injected with the bicistronic construct with the 59-UTR in the forward orientation
showed significant blue and green fluorescence. This indicates an IRES in the 59-UTR of ELH mRNA [43].
The next issue investigated was whether IRES activity
in the 59-UTR of ELH mRNA is regulated by the afterdischarge [43]. The BCNs in both clusters were injected with
the bicistronic construct with the 59-UTR in the forward
orientation. One cluster was stimulated to afterdischarge,
and the other was an unstimulated control. If afterdischarge
stimulates IRES activity, then the ratio of green:blue fluorescence should be greater in the stimulated neurons compared to that in the controls, and in fact, this was the case.
Afterdischarges stimulated a twofold increase in the ratio
of green:blue fluorescence compared to that of unstimulated
controls [43]. Coincidentally, this is the same magnitude of
increase observed for the afterdischarge-induced increase
in ELH biosynthesis. These results suggest that the afterdischarge stimulates ELH synthesis by activating an IRES
in the 59-UTR of ELH mRNA. Recent work involving
HeLa cells showed that dephosphorylation of eIF4E during
mitosis is associated with disruption of the eIF4F complex
and inhibition of cap-dependent translation [42]. In Aplysia
sensory neurons, injecting a plasmid encoding a nonphosphorylatable eIF4E mutant inhibited eIF4E phosphorylation
and caused a switch to cap-independent, IRES-mediated
translation [43]. Together, these findings are consistent with
the hypothesis that the afterdischarge dephosphorylates
eIF4E, which is responsible for a switch from cap-dependent translation to IRES-mediated translation of ELH
mRNA. Notably, this work described in BCNs is, to our
knowledge, the first evidence for a physiological use of an
IRES in neurons—and one of the few examples in any cell
type. Nevertheless, use of an IRES in cellular mRNAs likely is an important mechanism by which synthetic machinery can be directed toward critically important peptides/
proteins at the expense of nonessential proteins.
CONCLUSION
The Aplysia BCNs are an important and unique model
system for exploring issues related to cellular and molecular mechanisms that regulate activity-dependent neurohormone synthesis and secretion. All the BCN work described
in the present review was performed with intact nervous
tissue in which normal physiological functions were preserved [4]. Several key findings related to activity-dependent regulation of ELH biosynthesis were discussed. First,
the afterdischarge stimulates a rapid and robust increase in
ELH synthesis to replenish releasable supplies of hormone.
Second, this stimulation of ELH synthesis results from an
increase in the rate of translation of ELH mRNA rather than
from an increase in transcription of the ELH gene. Third,
the afterdischarge selectively stimulates ELH synthesis, but
total protein synthesis is inhibited, indicating that a unique
mechanism is used to control translation of a specific peptide. It was then shown that the afterdischarge dephosphorylates eIF4E and activates an IRES at the 59-UTR of
ELH mRNA, with changes in the phosphorylated state of
eIF4E potentially leading to a switch from cap-dependent
to cap-independent translation. This could explain the effects of afterdischarge on selectively stimulating ELH
mRNA translation while simultaneously inhibiting total
protein synthesis.
What is ultimately important to the animal is to be reproductively successful. Aplysia have a single breeding season during which to reproduce. In fact, their reproductive
system peaks in function at a time when many other physiological systems are breaking down. By using a unique
translational mechanism, the reproductive neuroendocrine
cells are able to funnel their synthetic machinery toward
the most important peptide—the one that leads to reproduction and passing on the animal’s genetic material.
CONTROL OF NEUROHORMONE SYNTHESIS
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