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First published online 18 October 2006
doi: 10.1242/dev.02619
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Department of Molecular, Cellular and Developmental Biology and Neuroscience Program, University of Michigan, 830 North University Avenue, Ann Arbor, MI 48109, USA.
* Author for correspondence (e-mail: makaabou{at}umich.edu)
Accepted 7 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Recycling, Receptor dynamics, Half-life, Phosphorylation, Synaptic activity
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid (AMPA) receptors
from intracellular endosomal compartments to the postsynaptic membrane
(Contractor and Heinemann,
2002
; Ehlers,
2000; Lee et al.,
2004; Luscher et al.,
1999; Park et al.,
2004). At the peripheral neuromuscular junction (NMJ), it was long
assumed that acetylcholine receptors (AChRs) remain stable in the postsynaptic
membrane until they are internalized and degraded in the lysosomes
(Gardner and Fambrough, 1979).
In this scheme, insertion of newly synthesized receptors would be responsible
for maintaining AChR density in the postsynaptic membrane over time. Recent
work from our laboratory has shown, however, that a significant number of
internalized AChRs are not degraded, but instead return back into the
postsynaptic membrane where they intermingle with AChRs that have not yet been
internalized and retain their initial fluorescent tag (referred to hereafter
as `preexisting' AChRs) (Bruneau et al.,
2005). However, the way these AChR pools are maintained and
regulated remains to be determined.
The involvement of phosphorylation in the trafficking, insertion and
maintenance of ionotropic receptors in central synapses has been extensively
studied, and has been found to play an important role in regulating synaptic
changes that lead to either long-term potentiation or long-term depression
(Carroll et al., 1999;
Ehlers, 2000;
Hayashi et al., 2000;
Lee et al., 2000;
Malinow and Malenka, 2002). At
the NMJ, a large body of work demonstrates that phosphatases are involved in
AChR anchoring and clustering in vivo and in vitro
(Grady et al., 2003;
Huganir et al., 1984;
Li et al., 2004;
Mei and Si, 1995;
Wallace, 1994;
Wallace et al., 1991);
however, the role of phosphorylation/dephosphorylation events in AChR
recycling at the NMJ in vivo has not previously been investigated.
In order to investigate the dynamics of recycled and pre-existing AChR
pools, and the potential role of phosphorylation/dephosphorylation in AChR
recycling, we monitored recycled and pre-existing AChRs using a sequential
labeling method that we had previously used to selectively identify recycled
and pre-existing receptor pools in the postsynaptic membrane
(Bruneau et al., 2005). Here,
we report that recycled receptors are removed more quickly than pre-existing
receptors from the same postsynaptic membrane of functional synapses, and
demonstrate that denervation decreases the insertion of recycled AChRs and
increases their removal rate. In addition, we found that tyrosine phosphatase
inhibitors, but not serine/threonine phosphatase inhibitors, cause the
aberrant perisynaptic accumulation of recycled AChRs.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lichtman et al., 1987;
van Mier and Lichtman, 1994).
Briefly, the anesthetized mouse was placed on its back on the stage of a
customized epifluorescence microscope, and NMJs were viewed under a coverslip
with water immersion objectives (20 x and 60 x UAPO 0.7 NA,
Olympus BW51, Optical Analysis Corporation, NH) and a digital CCD camera
(Retiga EXi, Burnaby, BC, Canada). Mice were intubated and ventilated for the
duration of the imaging sessions. For imaging at multiple time points, the
mouse was sutured after each session and allowed to fully recover before the
next imaging session.
Labeling of distinct AChR pools
The labeling protocol was performed as previously described
(Bruneau et al., 2005).
Briefly, the sternomastoid muscle was bathed first with fully substituted
bungarotoxin (BTX)-biotin (5 µg/ml, 1 hour; Molecular Probes, Eugene, OR)
to label AChRs, and then with streptavidin conjugated to Alexa 488
(strept-488; green; 10 µg/ml, 3 hours; Molecular Probes) to saturate all
biotin sites. To ensure that all biotin sites were saturated, a second,
differently-colored label, streptavidin-Alexa 594 (strept-594; red; 10
µg/ml, 10-20 minutes) was added to the sternomastoid muscle, and the
synapses imaged. The absence of strept-594 staining indicated that all biotin
sites were initially saturated with strept-488. Three to 4 days later, the
mouse was re-anesthetized, and the sternomastoid muscle re-exposed and bathed
with strept-594 to label the AChRs that had recycled back to the muscle
surface as AChR-BTX-biotin complexes. The muscle was washed out continuously
for 15 minutes with Ringer's solution. A brief chase of unlabeled streptavidin
(10 minutes) was added to the sternomastoid muscle to prevent the (unlikely)
binding to recycled AChRs of any residual fluorescent streptavidin that
remained in the milieu after the extensive washing. This labeling procedure
would prevent any erroneous estimation of receptor removal. The doubly labeled
superficial synapses were then imaged (IPLAB software, Scanalytics, VA). At
subsequent time points, the same synapses were relocated and imaged. In this
way, we were able to distinguish the pre-existing BTX-biotin-labeled receptors
(retaining their initial streptavidin) from the recycled receptor pools. It is
worth noting that at the time of recycled AChR labeling, a significant number
of receptors in the NMJ are new, functional receptors, and these are
sufficient to allow normal synaptic transmission
(Bruneau et al., 2005;
Lingle and Steinbach, 1988).
All controls for the specificity of biotin-streptavidin dissociation were
established previously by Bruneau et al.
(Bruneau et al., 2005).
To determine the loss rate of newly synthesized receptors, the sternomastoid muscle was incubated with unlabeled BTX (5 µg/ml, 1.5 hours) to saturate all surface receptors (a second dose of fluorescent BTX was used to verify that all AChRs were saturated). Four to 5 days after initial labeling, newly inserted receptors were labeled with BTX-biotin at a sub-saturating dose (5 µg/ml for 20 minutes), so that synaptic transmission remained functional, followed by a saturating dose of strept-488 (green). Superficial synapses were imaged and fluorescence loss was monitored over several days.
Surgical procedures
Sternomastoid muscles were denervated by excising a 5 mm piece of the
sternomastoid nerve to prevent re-innervation. To determine the effect of
denervation on the number of receptors recycled at the postsynaptic membrane,
mice denervated 6-8 days earlier were anesthetized and the sternomastoid
muscle was exposed and labelled first with BTX-biotin, and then with a single
saturating dose of strept-594, and superficial synapses were imaged as
described above. Three days later, the animal was reanaesthetized, the same
synapses were imaged and the loss of fluorescence measured. The sternomastoid
muscle was bathed again with fresh strept-594 (to label recycled AChRs),
synapses were imaged and the fluorescence intensity assayed. For comparison, a
similar labeling protocol was applied to innervated synapses.
To determine the removal rate of recycled and pre-existing AChRs in denervated muscle, the sternomastoid muscle of mice denervated 6-8 days earlier was labeled with BTX-biotin, followed by a single saturating dose of strept-488. Three days later, the sternomastoid muscle was exposed and synapses labeled with a single saturating dose of strept-594 (to label all receptors that had recycled over that time); then, superficial synapses were imaged, their fluorescence intensities were measured, and the decrease in fluorescence of both AChR pools was monitored over time.
To determine the effect of muscle action potentials on AChR turnover, immediately after recycled receptor labeling in denervated NMJs, the muscle was directly stimulated with a Grass SD5 stimulator connected to two platinum wires placed either side of the muscle. The stimulus pulses (3 msecond bipolar pulses of 6-9 V at 10 Hz for 1 second duration every 2 seconds) elicited maximal twitching and therefore action potentials in all muscle fibers. Mice were continuously ventilated and maintained under anesthesia by intraperitoneal injections of KX every 2 hours for the duration of the experiment. To minimize dehydration, a coverslip was placed over the exposed muscle.
Quantitative fluorescence imaging
The fluorescence intensity of labeled receptors at NMJs was assayed using a
quantitative fluorescence imaging technique, as described by Turney and
colleagues (Turney et al.,
1996), with minor modifications. This technique incorporates
compensation for image variation that may be caused by spatial and temporal
changes in the light source and camera between imaging sessions, by
calibrating the images with a non-fading reference standard. A key feature of
the quantitative imaging approach used in the current study is that it
involves repetitive imaging of the same fluorescent ligands (strept-594 and
strept-488). Thus, as long as we verified that labeling had reached saturation
and that the image pixel intensity was not saturated, it was relatively
trivial to get an accurate quantitative measurement of the relative number of
AChRs.
Pharmacology
For all agents tested, 500 µl of a stock solution was placed directly
onto the sternomastoid muscle for 9 hours. During the experiment, the animal
was intubated with a ventilator to avoid asphyxiation. To inhibit tyrosine
phosphatase activity, we used two agents: phenylarsine oxide (2 mM; Sigma, St
Louis, MO) and pervanadate (5-10 mM). Pervanadate solution was prepared by
mixing 1.7% H2O2 and sodium orthovanadate (Sigma) in a
ratio of 1:50 for 10 minutes before adding it to the sternomastoid muscle
(Madhavan et al., 2005).
Okadaic acid (10-100 µM; Sigma) was used to inhibit serine/threonine
phosphatases. To verify the effectiveness of okadaic acid in vivo, at the end
of the imaging session the sternomastoid muscle that had been bathed with
okadaic acid was removed from the mouse and homogenized in RIPA buffer
(Sigma). The supernatant was then subjected to a direct fluorescence-based
assay for detecting serine/threonine phosphatase activity (RediPlateTM 96
EnzChek serine/threonine phosphatases Assay Kit; Molecular Probes), according
to the manufacturer's instructions. Briefly, appropriate buffers for the
serine/threonine phosphatases PP1 and PP2A were added to a 96-well microplate
preloaded with inhibitors of phosphatases other than serine/threonine
phosphatases, and with the fluorogenic serine/threonine phosphate substrate
DiFMUP (6,8-difluoro-4-methyl-umbelliferyl phosphate), from which DiFMU is
generated. DiFMUP was then fully solubilized and homogenates from treated and
untreated muscle were added to the wells. The fluorescence emitted by DiFMU
was measured using a fluorescence microplate reader, with excitation at
355±20 nm and emission at 460±12.5 nm.
Confocal microscopy
The sternomastoid muscle was saturated with BTX-biotin followed by a single
saturating dose of strept-488 (10 µg/ml, 3 hours). Three to 4 days later,
the animal was re-anaesthetized and strept-594 was added to the sternomastoid
muscle to label recycled AChRs. Two to 3 days later, the animal was perfused
transcardially with 2% paraformaldehyde (PFA) and the sternomastoid muscle
removed and longitudinally sectioned. Muscle sections (20 µm) were blocked
with 10% BSA and 0.5% Triton X-100, and then bathed with monoclonal
anti-receptor antibody (mAb 35; Developmental Studies Hybridoma Bank, Iowa
University, IA), followed by an anti-rat secondary antibody conjugated to
Alexa 647 (Molecular Probes). Muscle sections were scanned with a confocal
microscope (Olympus fluoview) and imaged. The z-stacks were then
collapsed and the contrast adjusted with Photoshop to maximally resolve
intracellular fluorescent spots.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), and we
wanted to know whether recycled receptors are as stable in the synapse as
pre-existing receptors (receptors retaining their strept-488 after initial
labeling). To test this, the sternomastoid muscle of live mice was labeled
with BTX-biotin, followed by a single saturating dose of strept-488 to
saturate all biotin sites. To ensure that all biotin sites were labeled, the
muscle was exposed to the differently-colored strept-594. Images of
superficial synapses showed no staining with strept-594, indicating that all
biotin sites were saturated with strept-488. The animal was then allowed to
recover. Three to 4 days after the initial labeling, strept-594 was added to
label recycled receptors that had lost their strept-488 label but retained
BTX-biotin, and images were taken immediately (day 0). The dissociation of
streptavidin from AChR-BTX-biotin does not occur spontaneously on the muscle
surface, but rather only after the complex (AChR-BTX-biotin/strept-488) is
internalized (Bruneau et al.,
2005). Pre-existing AChRs that retain BTX-biotin/strept-488 after
the initial labeling and recycled AChRs labeled with BTX-biotin/strept-594
could then be imaged separately, and the changes in their fluorescence
intensities monitored over time. As shown in
Fig. 1A, recycled and
pre-existing AChRs intermingled in the same postsynaptic membrane. One to 3
days after labeling recycled AChRs with strept-594, both recycled and
pre-existing AChRs were re-imaged, once or multiple times, and then their
fluorescence intensities assayed. We found that after 1 day, the fluorescence
intensity of recycled receptors (receptors tagged with BTX-biotin/strept-594)
decreased to 59% (±5 s.d., n=25) of their original
fluorescence, which corresponds to a half-life of 
1.3 days. At 2 and 3
days, the remaining fluorescence intensities were 36% (±6 s.d.,
n=20) and 16% (±2 s.d., n=15) of the original
fluorescence, respectively. However, at the same synapses over the same times,
the fluorescence intensity of AChRs labeled with BTX-biotin/strept-488
decreased to only 84% (±6 s.d., n=20) of the original
fluorescence after 1 day (half-life 4 days), and after 2 and 3 days the
fluorescence had decreased to 68% (±9 s.d., n=10) and 54%
(±4 s.d., n=15), respectively
(Fig. 1B,C). Similar results
were obtained if strept-594 was used initially to label pre-existing receptors
and strept-488 to label recycled AChRs.
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Given the rapid loss of recycled receptors from the postsynaptic membrane, we next examined whether this loss is matched by the insertion of new recycled receptors over the same time period. The sternomastoid muscle of three mice was labeled with BTX-biotin followed by a saturating dose of strept-488. Three to 4 days later the animal was anaesthetized and the sternomastoid muscle bathed with a saturating dose of strept-594 to label the recycled receptors, as described above. The superficial synapses were then imaged, and the animal was allowed to recover. Two days later, the same synapses were re-imaged and the fluorescence of each synapse measured at each data point. The sternomastoid muscle was then bathed with a saturating dose of fresh strept-594 to label receptors that had been recycled over the given time period. When we measured the fluorescence intensity at 2 days, we found the fluorescence remaining from the recycled pool (labeled with strept-594) was 35% (±6 s.d., n=17) of the original fluorescence. When re-labeled with new strept-594, the fluorescence intensity returned to 99% (±17 s.d., n=10) of the initial recycled AChR fluorescence (Fig. 2A,B). This indicates that the insertion of new recycled receptors matches the loss of recycled receptors from the postsynaptic membrane.
Targeting of recycled AChRs to stable fluorescent intracellular vesicles after internalization
We have previously shown that receptors that are removed from synapses are
internalized in intracellular endocytic vesicles
(Akaaboune et al., 1999;
Bruneau et al., 2005). Because
recycled and pre-existing receptors intermingle at the same postsynaptic
membrane but turn over at such different rates
(Fig. 1), we wanted to know
whether recycled and pre-existing receptors, after their removal from the
postsynaptic membrane, are internalized in the same or in distinct
intracellular vesicles. To examine this, the sternomastoid muscle was labeled
with BTX-biotin/strept-488 (green), and 3-4 days later the recycled receptors
were labeled with strept-594 (red). When recycled and pre-existing AChRs were
reimaged 2 or more days later, we were surprised to find that all the vesicles
that contained red fluorescence from recycled receptors also contained green
fluorescence from pre-existing receptors (n=50 synapses;
Fig. 3A). This prompted us to
monitor the formation and accumulation of both green and red intracellular
puncta using time-lapse imaging. The sternomastoid muscle was labeled with
BTX-biotin/strept-488, and 3-4 days later the receptors that had recycled over
this time were labeled with strept-594. When superficial synapses were imaged
immediately after strept-594 (red) labeling, the AChRs labeled with green
strept-488 were concentrated in the postsynaptic membrane and in internal
compartments visualized as small spots of fluorescence in the vicinity of the
junction (Fig. 3B, top panels).
When the same synapse was imaged 8 hours later, some of the green spots
remained whereas others had disappeared. At this time point, faint red
fluorescent spots began to appear, but only at the stable spots of green
fluorescence (Fig. 3B, middle
panels). After 28 hours, although many of the green fluorescent spots had
disappeared, stable spots that contained both strept-488 and strept-594
remained (Fig. 3B, bottom
panels). These results indicate either that the removed recycled receptors
were directly and specifically targeted to stable vesicles already containing
internalized AChR-BTX-biotin/strept-488 complexes, or that the fluorescent
puncta observed in the intracellular vesicles were from streptavidin-Alexa
tags that had been removed from both receptor pools after internalization (see
Discussion).
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Next, we investigated whether recycled and pre-existing AChRs have similar
removal rates in denervated muscle. Junctions denervated 6-8 days earlier were
labeled with BTX-biotin/strept-488 and the animals allowed to recover. Three
days later, the recycled AChRs were labeled with strept-594, and superficial
synapses were imaged immediately and then re-imaged 1 and 2 days later. We
found that after only 1 day, the strept-594 fluorescence intensity (labeled
recycled AChRs) decreased dramatically to just 35% (± 5 s.d.,
n=17) of the original fluorescence (corresponding to a half-life of
15 hours, nearly twice as fast as the half-life of recycled AChRs at
innervated synapses). Loss of fluorescence continued, decreasing to 16%
(±3 s.d., n=9) after 2 days. At the same time, the strept-488
fluorescence from pre-existing receptors decreased to 70% (±6 s.d.,
n=10) after 1 day (half-life 1.9 days, also nearly two times
faster than the half-life at innervated synapses), and to 43% (±6 s.d.,
n=9) of their original fluorescence after 2 days
(Fig. 6A,B). These results
indicate that denervation increases the already rapid removal of recycled
receptors from the postsynaptic membrane, and has a similar effect on both
recycled and pre-existing AChRs, nearly doubling the rate of removal of each
receptor pool.
Because the above experiments indicate that muscle activity is important for receptor stability, we next tested whether direct muscle stimulation could prevent the rapid removal of recycled AChRs from the postsynaptic membrane. Denervated sternomastoid muscle was saturated (6-8 days after denervation) with BTX-biotin/strept-488, and then 3 days later was saturated with strept-594 to specifically label recycled AChRs. Superficial synapses were imaged and muscle action potentials were then elicited via stimulating electrodes placed at either end of the muscle (3 msecond bipolar pulses of 6-9 V at 10 Hz for 1 second every 2 seconds for the entire 8 hour period). For the duration of the experiment, the mouse was intubated and ventilated to prevent asphyxia. The result was dramatic: the loss of recycled AChRs observed in denervated muscles over the 8 hours (66% of original fluorescence remaining ±5 s.d., n=8) was almost completely prevented by the stimulation of denervated muscles (94% of original fluorescence remaining ±6 s.d., n=16) (Fig. 7A-C). This indicates that muscle action potentials are sufficient to prevent the loss of recycled receptors.
Proper localization of recycled receptors requires tyrosine phosphatase activity
Because phosphorylation and dephosphorylation events are associated with
postsynaptic receptor cycling in central synapses
(Ehlers, 2000;
Esteban et al., 2003;
Malinow and Malenka, 2002), we
next tested whether AChR recycling also depends upon
phosphorylation/dephosphorylation events. The sternomastoid muscle was labeled
with BTX-biotin/strept-488 (green), and the muscle then bathed continuously in
a phosphatase inhibitor. The mice were anesthetized and ventilated to prevent
asphyxia. After a 9-hour incubation with the drug, strept-594 (red) was added
to the sternomastoid muscle to label receptors that had been recycled back to
the membrane after initial labeling. We found a striking alteration in
receptor localization at synapses when the muscle was treated with
phenylarsine oxide (PAO), a known inhibitor of tyrosine phosphatases
(Fletcher et al., 1993;
Moult et al., 2006;
Moult et al., 2002). In
contrast to untreated control muscles, where the recycled surface receptors
were restricted to the junctional area as were the original receptors, PAO
treatment resulted in red labeling in the perisynaptic region as well as the
junctional region (Fig. 8B,C).
The presence of red signal in the peri-synaptic region is specific to recycled
receptors labeled with strept-594 (red), because at 9 hours no pre-existing
receptors (green) were detected outside of the original synapse
(Fig. 8A). Treatment with
another common tyrosine phosphatase inhibitor, pervanadate
(Madhavan et al., 2005;
Moult et al., 2006), caused
the same peri-synaptic staining of recycled AChRs
(Fig. 8E,F), without affecting
the synaptic localization of pre-existing AChRs
(Fig. 8D). These results
indicate that tyrosine phosphatase activity is involved in the correct
localization of recycled receptors.
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).
Nine hours later, when the muscle was labeled with strept-594, specific
staining was observed only in the junctional zone, indicating that the
targeting of recycled receptors does not require serine/threonine phosphatases
(Fig. 8G-I). The effectiveness
of this drug in vivo was tested at the end of the imaging session by removing
and homogenizing the sternomastoid muscle, and testing the activity of PP1 and
PP2A phosphatases using a serine/threonine phosphatase assay kit. When the
muscle was incubated in 10 µM okadaic acid, most of the serine/threonine
phosphatase activity (>70%) was inhibited, when compared with untreated
muscle. When 100 µM okadaic acid was used, all serine/threonine phosphatase
activity was inhibited and the muscle also showed signs of swelling.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An interesting finding of this work is that the lifetime of recycled
receptors is far shorter than that of the pre-existing AChRs, even though all
of these AChRs are intermingled in the same postsynaptic membrane. It is
unlikely that the difference in receptor lifetime obtained in our studies is
due to BTX binding itself, as we are comparing the removal rates of receptors
that are both tagged with BTX. However, it must be acknowledged that the use
of BTX, which specifically and irreversibly binds to AChRs, also blocks AChR
function. Although there is no direct evidence as to whether BTX binding
itself changes the behavior of AChRs, a number of previous studies suggest
that labeled receptors behave normally as long as NMJ activity is not totally
blocked (Akaaboune et al.,
1999; Lingle et al., 1988). Although it has been shown previously
that the gamma and epsilon subunits of AChRs are able to turn over at
different rates, it is unlikely that the differences in loss rates are due to
differences in the subunit composition of receptors (gamma versus epsilon)
because innervated adult synapses only express the epsilon subunit
(Burden, 1977;
Missias et al., 1996;
Yumoto et al., 2005). This
implies that an unknown mechanism exists that specifically alters recycled
AChR stability.
It is possible that during the process of intracellular receptor
trafficking (in early endosomes or specialized recycling endosomes),
alteration or modification of AChR subunits or receptor-associated proteins
may occur. Such alterations might affect the interaction of receptors with
postsynaptic scaffolding proteins, which in turn may affect their stability
once inserted into the postsynaptic membrane. For example, it has been
suggested that phosphorylation events are involved in the stabilization of
AChRs (Caroni et al., 1993),
and inhibition of these events has been shown to increase receptor removal and
prevent receptor clustering, despite ongoing muscle activity
(Ferns et al., 1996;
Fuhrer et al., 1997;
Wallace, 1994;
Wallace et al., 1991). At
central synapses, it has been shown that a coordination of phosphorylation and
dephosphorylation events may regulate synaptic strength
(Moult et al., 2006;
Raymond et al., 1993;
Raymond et al., 1994;
Roche et al., 1994); it is
possible that similar mechanisms may be involved at the NMJ. This possibility
is supported by our results, which show that inhibition of tyrosine
phosphatase activity can cause selective disruption of receptor recycling,
resulting in labeling outside of the usually sharp NMJ boundary, whereas
pre-existing receptors remain intact within the NMJ. This suggests that the
phosphorylation of receptors or associated proteins at tyrosine sites during
trafficking might cause a mistargeting of recycled receptors or increase their
mobility once they have recycled into the postsynaptic membrane. It is also
conceivable that tyrosine phosphatase inhibitors could selectively increase
the insertion and accumulation of recycled receptors at the peri-junctional
region. Interestingly, the same observation has been made previously in the
synapses of frog nerve/muscle co-cultures treated with tyrosine phosphatase
inhibitors, where is was attributed to the dissociation and migration of
pre-existing receptors into the peri-synaptic region
(Dai and Peng, 1998). Because
we were able to distinguish between pre-existing and recycled pools of
receptors, our results suggest that tyrosine phosphatase inhibition does not
cause dissociation of pre-existing receptors, but rather that it specifically
interferes with the delivery of internalized receptors back into the NMJ or
with their maintenance at the postsynaptic membrane.
Muscle activity appears to be crucial for the insertion and stability of
recycled AChRs in the postsynaptic membrane. Our results indicate that the
contribution of recycled AChRs is significantly depressed in denervated muscle
synapses when compared with innervated synapses. However, the inhibition of
recycling is not affected within the first week of denervation (data not
shown). It is possible that during the first week of muscle denervation, the
insertion of recycled receptors is maintained by spontaneous release of
acetylcholine neurotransmitter by Schwann cells
(Brett et al., 1982;
Dennis and Miledi, 1974),
which might explain why the number of synaptic receptors only begins to
decline 9 days after muscle denervation
(Andreose et al., 1995).
Consistent with this proposal, we have previously shown that spontaneous
release of a neurotransmitter is sufficient to prevent the loss of receptors
from synapses (Akaaboune et al.,
1999). This suggests that the decrease in receptor number at
denervated muscle synapses over time could be attributed to a decrease in the
delivery of the recycled receptor pool back to the postsynaptic membrane.
Interestingly, the absence of muscle activity following denervation not only
increases the removal rate of pre-existing AChRs, but also accelerates the
already rapid removal of the receptors that have recycled back into the
membrane. This indicates that recycled receptors, despite their rapid loss,
can still be controlled by synaptic activity. In agreement with our previous
findings, which showed that the stimulation of denervated muscle prevents the
removal of pre-existing AChRs (Bruneau et
al., 2005), in the present study we found that muscle stimulation
also prevents the removal of recycled receptors. Consistent with these
results, muscle stimulation alone has been reported to reversibly increase the
metabolic stability of receptors at synapses that are denervated early in
development, and can prevent the decrease in AChR half-life observed at
surgically denervated and blocked endplates
(Akaaboune et al., 1999;
Brenner and Rudin, 1989;
Rotzler and Brenner, 1990).
However, the way in which neuromuscular transmission or muscle action
potentials regulate AChR behavior is not understood. It is possible that
muscle contraction caused by direct stimulation causes an increase in calcium
influx through either ligand-gated or L-type channels, or that it causes
calcium release directly from intracellular stores. In support of this idea,
previous studies have shown that blockade of L-type channels affects AChR
stability (Rotzler et al.,
1991).
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Akaaboune, M., Culican, S. M., Turney, S. G. and Lichtman, J.
W. (1999). Rapid and reversible effects of activity on
acetylcholine receptor density at the neuromuscular junction in vivo.
Science 286,503
-507.
Andreose, J. S., Fumagalli, G. and Lomo, T. (1995). Number of junctional acetylcholine receptors: control by neural and muscular influences in the rat. J. Physiol. 483,397 -406.
Brenner, H. R. and Rudin, W. (1989). On the effect of muscle activity on the endplate membrane in denervated mouse muscle. J. Physiol. 410,501 -512.
Brett, R. S., Younkin, S. G., Konieczkowski, M. and Slugg, R. M. (1982). Accelerated degradation of junctional acetylcholine receptor-alpha-bungarotoxin complexes in denervated rat diaphragm. Brain Res. 233,133 -142.[CrossRef][Medline]
Bruneau, E., Sutter, D., Hume, R. I. and Akaaboune, M.
(2005). Identification of nicotinic acetylcholine receptor
recycling and its role in maintaining receptor density at the neuromuscular
junction in vivo. J. Neurosci.
25,9949
-9959.
Burden, S. (1977). Acetylcholine receptors at the neuromuscular junction: developmental change in receptor turnover. Dev. Biol. 61,79 -85.[CrossRef][Medline]
Caroni, P., Rotzler, S., Britt, J. C. and Brenner, H. R. (1993). Calcium influx and protein phosphorylation mediate the metabolic stabilization of synaptic acetylcholine receptors in muscle. J. Neurosci. 13,1315 -1325.[Abstract]
Carroll, R. C., Lissin, D. V., von Zastrow, M., Nicoll, R. A. and Malenka, R. C. (1999). Rapid redistribution of glutamate receptors contributes to long-term depression in hippocampal cultures. Nat. Neurosci. 2,454 -460.[CrossRef][Medline]
Contractor, A. and Heinemann, S. F. (2002). Glutamate receptor trafficking in synaptic plasticity. Sci. STKE 2002,RE14 .[Medline]
Dai, Z. and Peng, H. B. (1998). A role of
tyrosine phosphatase in acetylcholine receptor cluster dispersal and
formation. J. Cell Biol.
141,1613
-1624.
Dennis, M. J. and Miledi, R. (1974).
Electrically induced release of acetylcholine from denervated Schwann cells.
J. Physiol. 237,431
-452.
Ehlers, M. D. (2000). Reinsertion or degradation of AMPA receptors determined by activity-dependent endocytic sorting. Neuron 28,511 -525.[CrossRef][Medline]
Esteban, J. A., Shi, S. H., Wilson, C., Nuriya, M., Huganir, R. L. and Malinow, R. (2003). PKA phosphorylation of AMPA receptor subunits controls synaptic trafficking underlying plasticity. Nat. Neurosci 6,136 -143.[CrossRef][Medline]
Ferns, M., Deiner, M. and Hall, Z. (1996).
Agrin-induced acetylcholine receptor clustering in mammalian muscle requires
tyrosine phosphorylation. J. Cell Biol.
132,937
-944.
Fletcher, M. C., Samelson, L. E. and June, C. H.
(1993). Complex effects of phenylarsine oxide in T cells.
Induction of tyrosine phosphorylation and calcium mobilization independent of
CD45 expression. J. Biol. Chem.
268,23697
-23703.
Fuhrer, C., Sugiyama, J. E., Taylor, R. G. and Hall, Z. W. (1997). Association of muscle-specific kinase MuSK with the acetylcholine receptor in mammalian muscle. EMBO J. 16,4951 -4960.[CrossRef][Medline]
Gardner, J. M. and Fambrough, D. M. (1979). Acetylcholine receptor degradation measured by density labeling: effects of cholinergic ligands and evidence against recycling. Cell 16,661 -674.[CrossRef][Medline]
Grady, R. M., Akaaboune, M., Cohen, A. L., Maimone, M. M.,
Lichtman, J. W. and Sanes, J. R. (2003).
Tyrosine-phosphorylated and nonphosphorylated isoforms of alphadystrobrevin:
roles in skeletal muscle and its neuromuscular and myotendinous junctions.
J. Cell Biol. 160,741
-752.
Hayashi, Y., Shi, S. H., Esteban, J. A., Piccini, A., Poncer, J.
C. and Malinow, R. (2000). Driving AMPA receptors into
synapses by LTP and CaMKII: requirement for GluR1 and PDZ domain interaction.
Science 287,2262
-2267.
Huganir, R. L., Miles, K. and Greengard, P.
(1984). Phosphorylation of the nicotinic acetylcholine receptor
by an endogenous tyrosine-specific protein kinase. Proc. Natl.
Acad. Sci. USA 81,6968
-6972.
Lee, H. K., Barbarosie, M., Kameyama, K., Bear, M. F. and Huganir, R. L. (2000). Regulation of distinct AMPA receptor phosphorylation sites during bidirectional synaptic plasticity. Nature 405,955 -959.[CrossRef][Medline]
Lee, S. H., Simonetta, A. and Sheng, M. (2004). Subunit rules governing the sorting of internalized AMPA receptors in hippocampal neurons. Neuron 43,221 -236.[CrossRef][Medline]
Li, M. X., Jia, M., Yang, L. X., Jiang, H., Lanuza, M. A.,
Gonzalez, C. M. and Nelson, P. G. (2004). The role of the
theta isoform of protein kinase C (PKC) in activity-dependent synapse
elimination: evidence from the PKC theta knock-out mouse in vivo and in vitro.
J. Neurosci. 24,3762
-3769.
Lichtman, J. W., Magrassi, L. and Purves, D. (1987). Visualization of neuromuscular junctions over periods of several months in living mice. J. Neurosci. 7,1215 -1222.[Abstract]
Lingle, C. J. and Steinbach, J. H. (1988). Neuromuscular blocking agents. Int. Anesthesiol. Clin. 26,288 -301.[Medline]
Luscher, C., Xia, H., Beattie, E. C., Carroll, R. C., von Zastrow, M., Malenka, R. C. and Nicoll, R. A. (1999). Role of AMPA receptor cycling in synaptic transmission and plasticity. Neuron 24,649 -658.[CrossRef][Medline]
Madhavan, R., Zhao, X. T., Ruegg, M. A. and Peng, H. B. (2005). Tyrosine phosphatase regulation of MuSK-dependent acetylcholine receptor clustering. Mol. Cell. Neurosci. 28,403 -416.[CrossRef][Medline]
Malinow, R. and Malenka, R. C. (2002). AMPA receptor trafficking and synaptic plasticity. Annu. Rev. Neurosci. 25,103 -126.[CrossRef][Medline]
Mei, L. and Si, J. (1995). Tyrosine phosphorylation and synapse formation at the neuromuscular junction. Life Sci. 57,1459 -1466.[CrossRef][Medline]
Missias, A. C., Chu, G. C., Klocke, B. J., Sanes, J. R. and Merlie, J. P. (1996). Maturation of the acetylcholine receptor in skeletal muscle: regulation of the AChR gamma-to-epsilon switch. Dev. Biol. 179,223 -238.[CrossRef][Medline]
Moult, P. R., Schnabel, R., Kilpatrick, I. C., Bashir, Z. I. and Collingridge, G. L. (2002). Tyrosine dephosphorylation underlies DHPG-induced LTD. Neuropharmacology 43,175 -180.[CrossRef][Medline]
Moult, P. R., Gladding, C. M., Sanderson, T. M., Fitzjohn, S.
M., Bashir, Z. I., Molnar, E. and Collingridge, G. L. (2006).
Tyrosine phosphatases regulate AMPA receptor trafficking during metabotropic
glutamate receptor-mediated long-term depression. J.
Neurosci. 26,2544
-2554.
Pahan, K., Sheikh, F. G., Namboodiri, A. M. and Singh, I.
(1998). Inhibitors of protein phosphatase 1 and 2A differentially
regulate the expression of inducible nitric-oxide synthase in rat astrocytes
and macrophages. J. Biol. Chem.
273,12219
-12226.
Park, M., Penick, E. C., Edwards, J. G., Kauer, J. A. and
Ehlers, M. D. (2004). Recycling endosomes supply AMPA
receptors for LTP. Science
305,1972
-1975.
Raymond, L. A., Blackstone, C. D. and Huganir, R. L. (1993). Phosphorylation and modulation of recombinant GluR6 glutamate receptors by cAMP-dependent protein kinase. Nature 361,637 -641.[CrossRef][Medline]
Raymond, L. A., Tingley, W. G., Blackstone, C. D., Roche, K. W. and Huganir, R. L. (1994). Glutamate receptor modulation by protein phosphorylation. J. Physiol. Paris 88,181 -192.[CrossRef][Medline]
Roche, K. W., Tingley, W. G. and Huganir, R. L. (1994). Glutamate receptor phosphorylation and synaptic plasticity. Curr. Opin. Neurobiol. 4, 383-388.[CrossRef][Medline]
Rotzler, S. and Brenner, H. R. (1990).
Metabolic stabilization of acetylcholine receptors in vertebrate neuromuscular
junction by muscle activity. J. Cell Biol.
111,655
-661.
Rotzler, S., Schramek, H. and Brenner, H. R. (1991). Metabolic stabilization of endplate acetylcholine receptors regulated by Ca2+ influx associated with muscle activity. Nature 349,337 -339.[CrossRef][Medline]
Turney, S. G., Culican, S. M. and Lichtman, J. W. (1996). A quantitative fluorescence-imaging technique for studying acetylcholine receptor turnover at neuromuscular junctions in living animals. J. Neurosci. Methods 64,199 -208.[CrossRef][Medline]
van Mier, P. and Lichtman, J. W. (1994). Regenerating muscle fibers induce directional sprouting from nearby nerve terminals: studies in living mice. J. Neurosci. 14,5672 -5686.[Abstract]
Wallace, B. G. (1994). Staurosporine inhibits
agrin-induced acetylcholine receptor phosphorylation and aggregation.
J. Cell Biol. 125,661
-668.
Wallace, B. G., Qu, Z. and Huganir, R. L. (1991). Agrin induces phosphorylation of the nicotinic acetylcholine receptor. Neuron 6, 869-878.[CrossRef][Medline]
Yumoto, N., Wakatsuki, S. and Sehara-Fujisawa, A. (2005). The acetylcholine receptor gamma-to-epsilon switch occurs in individual endplates. Biochem. Biophys. Res. Commun. 331,1522 -1527.[CrossRef][Medline]
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