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Vol. 18, Issue 8, 2904-2911, August 2007
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Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Program, University of Michigan, Ann Arbor, MI 48109
Submitted February 2, 2007;
Revised April 9, 2007;
Accepted May 17, 2007
Monitoring Editor: Tom U. Martin
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dystrobrevin, a component of the dystrophin glycoprotein complex, we found that the density and distribution of synaptic AChEs are profoundly altered and that the loss rate of AChE significantly increased. These results demonstrate that nonsynaptic AChEs are mobile, whereas synaptic AChEs are more stable, and that -dystrobrevin is important for controlling the density and stability of AChEs at neuromuscular synapses. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Soreq and Seidman, 2001). The efficacy by which AChE hydrolyzes acetylcholine depends not only on its location but also on the number and density of AChE at synaptic sites. Indeed, reduction in the number and density of AChE leads to failures of neuromuscular transmission. For example, patients with myasthenia syndrome or mice lacking the collagen tailed protein ColQ, perlecan, or dystroglycan (Ohno et al., 1998; Feng et al., 1999; Jacobson et al., 2001; Arikawa-Hirasawa et al., 2002) fail to concentrate AChEs at the neuromuscular junction (NMJ), greatly altering synaptic transmission.
At adult sternomastoid NMJs, the density of AChE is estimated to be >3000 molecules/µm2, composed predominantly of the asymmetric A12 form (Salpeter et al., 1972, 1979; Massoulie et al., 1993; Anglister et al., 1994, 1998). In developing synapses, AChEs are diffusely distributed all along the muscle fiber (Sketelj and Brzin, 1980; Koenig and Rieger, 1981; Fernandez and Seiter, 1984), and they become aggregated at nerve contact sites as synapses mature. Until now, however, it has not been possible to study the mobility of AChE at functioning adult synapses in vivo; therefore, it is not known whether there is a continuous exchange of AChE between nonsynaptic and synaptic zones.
Although the role of the dystrophin glycoprotein complex (DGC) in the maintenance of the postsynaptic receptor density at the neuromuscular junction has been extensively studied (Straub and Campbell, 1997; Grady et al., 2000; Akaaboune et al., 2002; Burton and Davies, 2002), little is known about the role of this complex on AChE dynamics in the synaptic cleft. In this work, we focused on -dystrobrevin, a DGC component that has been shown to play a critical role in the maturation and the maintenance of AChR density and turnover at synapses (Grady et al., 2000; Akaaboune et al., 2002). Because the ratio of AChE to AChR seems to be critical for normal functioning synapses, we sought to investigate the behavior of AChE in synapses deficient in -dystrobrevin.
Using in vivo fluorescence imaging assays and fasciculin2 snake toxin, which binds selectively to AChE (Martinez-Pena y Valenzuela et al., 2005; Krejci et al., 2006), we investigated AChE dynamics at individual synapses over time. In the first part of this work, we examined the mobility of synaptic and nonsynaptic AChEs, and we found that nonsynaptic AChEs are free to move on the surface of the muscle fiber and to contribute to synaptic density, whereas synaptic AChEs are immobile. In the second part of this work, we studied AChE dynamics at synapses lacking -dystrobrevin, and we found that the density and turnover of AChEs are dramatically affected.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dystrobrevin–deficient mice (10 wk old; 129SVJ x C57/BL6 hybrid background; Grady et al., 2000) were generously provided by Dr. Joshua Sanes (Harvard University), and they were bred in our animal facility. All animal use followed methods approved by the University of Michigan Committee on the Use and Care of Animals.
Toxins
Unlabeled fasciculin2 was purchased from Latoxan (Valence, France), and it was conjugated to biotin using the Biotin-XX Microscale Protein Labeling kit (Invitrogen, Carlsbad, CA). Conjugated bungarotoxin was purchased from Invitrogen. For in vivo imaging of the neuromuscular junction, the mice were anesthetized with an intraperitoneal (i.p.) injection of a mixture of ketamine and xylazine (KX) (17.38 mg/ml). Sternomastoid muscle exposure and neuromuscular junction imaging were done as described in detail previously (Lichtman et al., 1987; van Mier and Lichtman, 1994; Akaaboune et al., 1999). Briefly, the anesthetized mouse was placed on its back on the stage of a customized fluorescence microscope, and neuromuscular junctions were viewed under a coverslip with a water immersion objective (20x UAPO 0.7 numerical aperture Olympus BW51; Optical Analysis, Nashua, NH) and digital charge-coupled device camera (Retiga EXi, Burnaby, British Columbia, Canada).
Quantitative Fluorescence Imaging
The fluorescence intensity of labeled AChEs at neuromuscular junctions was assayed using a quantitative fluorescence imaging technique, as described by Turney et al. (1996), with minor modification. 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 at each imaging session with a nonfading reference standard. Image analysis was performed using either a procedure written for IPLab (Scanalytics, Fairfax, VA) or MatLab (Mathworks, Natick, MA). Background fluorescence was approximated by selecting a boundary region around the junction and subtracting it from the original image. The sum of the fluorescent of AChEs at a later time was expressed as the percentage of the sum of fluorescence of AChEs at time 0, which was set at 100%. Average intensity is presented as mean ± SD.
Fluorescence Recovery after Photobleaching Experiments
To investigate the contribution of nonsynaptic (perisynaptic and extrasynaptic) AChEs to the synapse, an argon laser attached to the microscope was used to selectively remove the fluorescence from all synaptically localized AChEs by carefully tracking the branches of the whole neuromuscular junction. The laser illumination was done in the presence of a high concentration of unlabeled streptavidin to prevent the rebinding of unbound fluorescent streptavidin due to photo-unbinding (Akaaboune et al., 2002; Akaaboune, Turney, and Lichtman unpublished data). In this way, only nonsynaptic, which include extrasynaptic and perisynaptic, AChEs remained labeled. Acquisition of a second image was used to confirm the removal of fluorescence. From 1–5 d later, the same synapses were reimaged one or multiple times, and the recovery of fluorescence into the bleached area was measured. To study the mobility of AChE, we used fasciculin2-biotin labeling followed by incubation with fluorescent streptavidin conjugate. Fasciculin2-biotin/streptavidin is ideal for this work, because its dissociation from AChE is negligible, and the removal rate of AChE labeled with either fasciculin2-biotin/streptavidin-Alexa or fasciculin2-Alexa is nearly the same. Finally, and most importantly, fasciculin2-biotin/streptavidin is not displaced by unlabeled fasciculin2 (Krejci et al., 2006).
To study the contribution of only extrasynaptic AChEs that migrate to the synaptic pool, a laser beam was used to bleach fluorescent conjugates bound to AChEs from synapses and their immediate vicinity 
100 µm away from the synapse along the length of the muscle (Salpeter et al., 1988), and a second image was used to confirm the removal of fluorescence. The wound was sutured and the animal was returned to its cage. The same NMJs were then relocated and imaged at subsequent views 2 and 3 d later.
To quantitate the insertion of newly synthesized AChEs, the sternomastoid muscle was saturated with fasciculin2-biotin/streptavidin Alexa 488, and superficial synapses were imaged. An argon laser was used to remove the fluorescence from the synaptic zone, and a second image was taken to confirm the absence of fluorescence. Three days later, the animal was anesthetized, and the same junctions were reimaged and a second dose of fasciculin2-biotin/streptavidin 488 was added to label AChEs that were inserted during this period of time, and images of the same synapses were taken again.
-Dystrobrevin Mutant Mice
Measurement of AChE Density.
To estimate the density of AChEs, the sternomastoid muscle of both adbn–/– mutant and wild-type mice were saturated with either fasciculin2-Alexa 594 (7 µg/ml; 3 h), or fasciculin2-biotin (7 µg/ml; 3 h) followed by a saturating dose of streptavidin-Alexa 488/594 (10 µg/ml; 3 h). The superficial neuromuscular junctions were imaged, and the mean fluorescence was determined using a quantitative fluorescence assay.
Loss and Insertion Rates of Fluorescent Labeled AChEs.
To determine the loss rate of AChE from wild-type and adbn–/– mutant synapses, the sternomastoid muscle was labeled with a low dose of fasciculin2-biotin (5 µg/ml; 30 min) (usually <40% of AChEs are labeled) followed by a saturating dose of streptavidin-Alexa 488. Usually, the first views of superficial synapses were taken after 1 d of initial labeling, to allow the clearance of unbound fasciculin2-biotin. On subsequent days, the same synapses were located and reimaged one or multiple times, and their fluorescence intensities were assayed. To determine the insertion rate of AChE in -dystrobrevin mutant mice, the sternomastoid muscle was saturated with fasciculin2-biotin/streptavidin-Alexa 488 and imaged. Twenty-four hours later, the synapses were imaged, saturated with fresh fasciculin2-biotin/streptavidin-Alexa 488, and reimaged.
Photo-Unbinding.
We used this technique to follow exclusively the movement of synaptic AChEs within single synapses from the same muscle fiber. This method allows the labeling of different populations of AChEs at the same NMJ with distinct fluorophores. The sternomastoid muscle was labeled with a low dose of fasciculin2-biotin (so synaptic activity remains functional) followed by a saturating dose of streptavidin-Alexa 488. Photo-unbinding was then performed as described by Akaaboune et al. (2002). Briefly, an argon laser (488 nm), 1–2 mW at the back aperture of the objective, was used to excite fluorescently labeled fasciculin2-biotin/streptavidin-Alexa 488 until all fluorescence was removed. Immediately, the sternomastoid muscle was incubated in the presence of saturating dose of a different color of streptavidin conjugated to Alexa 594 to selectively label the laser-illuminated region. The doubly labeled junctions were imaged, and the wound was then sutured. The same synapses were reimaged again 1–2 d later.
Confocal Imaging.
To determine the distribution of AChE at the synaptic cleft, the sternomastoid muscles of both wild-type and mutant synapses were labeled with fluorescent -bungarotoxin-Alexa 594 and fasciculin2-Alexa 488, and then they were perfused with 4% paraformaldehyde. The sternomastoid muscles were dissected and mounted on slides, and then they were scanned using confocal microscopy (Olympus FV500; Olympus America, Melville, NY).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Akaaboune, Turney, Sanes, and Lichtman, unpublished data). We then monitored the recovery of fluorescence at bleached junctions over time. We found that over the next 24 h, junctions gained 2.4 ± 0.7% (mean ± SD; n = 32) of their original fluorescence (Figure 1C). The intensity of fluorescence recovery gradually increased to reach 4 ± 1% (n = 19) and 5 ± 1.2% (n = 27) at 48 and 72 h, respectively (Figure 1). At 5 d, the fluorescence recovery at bleached synapses, however, declined to 2.2 ± 1% (n = 10). When synapses were labeled and not photobleached, they lost 17 ± 8% (n = 51) of their fluorescence intensity per day (Figure 4). Thus, the fluorescence recovery observed after photobleaching suggests that a significant number of nonsynaptic AChEs migrate into the synaptic zone (see Discussion). To rule out the possibility of that the recovery of fluorescence observed in living muscle was due to reversible bleaching of the fluorophore or to the rebinding of labeled toxin, a sternomastoid muscle was first fixed with 2% paraformaldehyde, labeled with fasciculin2-biotin/streptavidin-Alexa 488, illuminated with laser to remove fluorescence, and then transplanted into the neck of a host mouse directly next to the living sternomastoid muscle. No recovery of original fluorescence into bleached synapses was observed over time (Figure 1B).
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4 and 5% when only fluorescently tagged junctional AChEs were bleached) (Figure 1D). This result implies that a significant amount of fluorescence recovery is due to the migration of AChEs that are located within 100 µm of the synaptic area. and it suggests that perisynaptic AChEs play a role in maintaining synaptic AChE density. We next wanted to determine the amount of AChEs that have been inserted directly into synaptic sites. To do this, the sternomastoid muscle was bathed with a saturating dose of fasciculin2-biotin/streptavidin-Alexa 488 to label both nonsynaptic and synaptic AChEs. The superficial synapses were then imaged, laser illuminated, and reimaged to confirm complete photobleaching of the synapses. Three days later, the same synapses were relocated and reimaged, and then saturating doses of fasciculin2-biotin and streptavidin-Alexa 488 were added to label newly inserted AChEs. We found that the fluorescence in synapses increased significantly to reach 55 ± 7% (n = 6) of original fluorescence (Figure 1E). These results support the idea that the majority of AChEs are inserted directly into synaptic sites.
Synaptic AChEs Are Immobile within the Neuromuscular Junction
Having found that nonsynaptic AChEs can move into synaptic sites, we next asked whether synaptic AChEs can migrate from one region to another within the same synapse. To assay the mobility of synaptic AChEs without contamination from nonsynaptic AChEs, the sternomastoid muscle was incubated with fasciculin2-biotin (labeling both synaptic and nonsynaptic AChEs), and biotin sites were saturated with streptavidin-Alexa 488. An argon laser was then used to selectively unbind green streptavidin-labeled AChEs from a small region within a junction, which was then relabeled with streptavidin Alexa 594 (red), as described previously (Akaaboune et al., 2002). When mobility of the red-labeled synaptic AChEs was monitored after 1 or 2 d after the initial labeling, we found that the red-labeled AChEs did not spread from their initial region into the rest of the junction, despite the overall loss of red fluorescence. Green fluorescence, however, was seen in the bleached region (Figure 2). This indicates that synaptic AChEs are almost entirely immobile within the synaptic zone, at least during the window of our experiments (see Discussion). and it implies that the removal of synaptic AChEs occurs locally.
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-Dystrobrevin-dystrobrevin dramatically affects the density and the number of postsynaptic AChRs (Grady et al., 2000, 2003; Akaaboune et al., 2002). Because the ratio of AChE to AChR is critical for normal synaptic functioning, we wanted to examine whether AChE density and turnover rate are also affected by the loss of -dystrobrevin. First, we wanted to determine the relative distribution of AChEs to AChRs in mutant mice lacking -dystrobrevin (adbn–/–). To do this, AChRs and AChEs at the sternomastoid muscle were both labeled with distinctive fluorophore-tagged toxins (-bungarotoxin-Alexa 594 for AChRs and fasciculin2-Alexa 488 for AChEs). Mice were then perfused with 4% paraformaldehyde, and sternomastoid muscles were dissected and mounted on slides and then scanned using confocal microscopy. In wild-type synapses, AChE staining extended beyond the AChR staining (Figure 3A), as reported previously (Adams et al., 2000; Krejci et al., 2006). However, in adbn–/– synapses, AChE staining was disorganized and resembled AChR distribution, which displays a granular appearance with frequent speckles of AChRs. AChE staining was absent from these speckles, probably because the folds are absent in such areas (Grady et al., 2000). This observation indicates that despite the intracellular localization of adbn, it is involved in the clustering and organization not only of AChRs but also of AChEs at the synapse (Figure 3, A–F).
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30% of those in normal junctions, whereas the overall size of synapses in mutant mice remained similar to that of wild type (Figure 3). This result suggests that -dystrobrevin plays an important role in maintaining the density of AChE at the synapse. AChRs are also reduced by similar amounts in muscles of the adbn–/– mice (Akaaboune et al., 2002), suggesting that AChE and AChR are present in an equal ratio at wild-type and adbn–/– synapses.
We asked whether the low density of AChE observed in adbn–/– synapses is associated with a rapid removal of AChE from the synapse or with a failure to properly insert AChE into the basal lamina. The rate of AChE removal from adbn–/– synapses was examined by labeling the sternomastoid muscle of mutant and wild-type mice with fasciculin2-biotin/streptavidin-Alexa 488 and by imaging superficial synapses. The fluorescence intensity of superficial neuromuscular junctions was measured at time 0, and then 1, 2, and 3 d later. We found that after initial labeling, AChE fluorescence intensity was decreased by 35 ± 10% (n = 59) at 1 d and by 49 ± 10% (n = 52) and 55 ± 9% (n = 27) at 2 and 3 d, respectively. At wild-type synapses, however, the loss was only 17 ± 8 (n = 51) at 1 d, 29 ± 8% (n = 54) at 2 d, and 34 ± 9% (n = 43) at 3 d (Figure 4, A–C). This rapid loss rate suggests that adbn is involved in controlling the lifetime of AChEs in the synaptic cleft. We next asked whether the low density of AChEs observed in mutant synapses is a consequence of a decreased insertion of newly synthesized AChEs, knowing that the shape of the synapses of these mutants remains constant at least over the time course of the experiments. To do this, the sternomastoid muscle of adbn–/– mice was saturated with fasciculin2-biotin followed with a saturating dose of streptavidin-Alexa 488, and then superficial synapses were imaged. When synapses were reimaged on subsequent days, we found that AChE insertion (determined when new fasciculin2-biotin/streptavidin-Alexa 488 was added) nearly matched AChE loss (Figure 4D). These results argue that despite the accelerated rate of AChE loss, the total number of AChEs was maintained by AChE insertion. To determine whether the increased rate of AChE loss in adbn–/– is due to a disassembly of anchoring molecules dystroglycan and/or perlecan, we performed immunostaining on the sternomastoid muscle with antibodies to dystroglycan and perlecan. We found that the distribution of these anchoring proteins in adbn–/– muscle are not altered (data not shown), as has been shown previously (Grady et al., 1999).
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-dystrobrevin from the postsynaptic membrane in mutant synapses. To investigate this, the sternomastoid muscle was labeled with a low dose of fasciculin2-biotin followed by a single saturating dose of streptavidin-Alexa 488 (as described above), and then superficial synapses were imaged. The synapses were laser illuminated to remove fluorescence from the NMJ, and they were immediately imaged to confirm complete bleaching. At 3 d, when maximum recovery was observed at wild-type synapses, adbn–/– synapses showed very little fluorescence recovery (2.4 ± 0.5%; n = 20); and in many cases. the fluorescence intensity was so low that it was very difficult to accurately quantify (Figure 4E). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-dystrobrevin, a component of the dystrophin glycoprotein complex, controls the density, distribution, and the lifetime of extracellular AChEs at synaptic sites.
Although it is not clear how diffusion of AChE in the nonsynaptic regions occurs, this work unambiguously shows that AChE can move from nonsynaptic to synaptic areas on the living muscle fiber. Given the high density of AChE in the synaptic cleft (3000 AChEs/µm2) (Salpeter et al., 1978; Anglister et al., 1994, 1998) and its high rate of removal from synapses (17%/d), the nonsynaptic AChEs may contribute to maintaining the high density of synaptic AChE despite the fact that the majority of AChEs are directly inserted into synaptic sites. Because AChE has been shown to not recycle back to the synaptic cleft (Bruneau et al., 2005), it is likely that insertion of AChE followed by diffusion and trapping of AChE in the NMJ is the main mechanism by which nonsynaptic AChE contributes to AChE synaptic density. Consistent with this, it has been shown that either endogenously deposited or experimentally transplanted AChE can be recruited and aggregated at the postsynaptic membrane (Rotundo et al., 1997). In developing muscle fibers, AChEs are found to be distributed diffusely over the muscle fiber, and they aggregate progressively into the synaptic zone as synapses mature (Sketelj and Brzin, 1980; Koenig and Rieger, 1981; Fernandez and Seiter, 1984; Legay et al., 1995). A similar developmental pattern has been described for AChRs (Anderson and Cohen, 1977; Ziskind-Conhaim et al., 1984). Lateral movement of AChE was also previously reported in cultured muscle cells (Peng et al., 1999). That both AChEs and AChRs are distributed diffusely along the muscle fiber surface, subsequently become aggregated at nerve muscle contacts, and migrate from nonsynaptic to synaptic zones may suggest that they share similar regulatory mechanisms. A constant ratio of AChE to AChR may be critical to ensure a normal, stable and effective physiological response at neuromuscular synapses.
One question our data raises is how AChE movement on the muscle surface may occur. Extensive studies have shown that AChE is anchored in the extracellular matrix through the collagen protein ColQ (Krejci et al., 1997), which in turn forms complexes with different acceptor molecules (Rotundo, 2003; Rotundo et al., 2005). Thus, it is possible that the diffusion trap mechanism observed on living muscle could be the result of the migration of AChE–ColQ complexes. If this is the case, it would suggest that the ColQ–AChE complex is highly dynamic despite its large size and multiple interactions with other proteins, and it is possible that other components of the extracellular matrix are also dynamic throughout the lifetime of a synapse. Biochemical analyses of the sedimentation patterns of muscle extracts have shown that AChE forms are expressed differentially in fast and slow twitch muscles. For example, in the mature sternomastoid muscle (fast muscle), A12 is the predominant form of AChE found at end plates, whereas A4 and A8 forms are present in the nonsynaptic zone of the soleus (slow muscle) (Krejci et al., 1999). It is conceivable that the sternomastoid muscle also produces a small quantity of A8 and A4 isoforms in its nonsynaptic segments while directly inserting A12 into the endplate. Alternatively, it is possible that the recovery seen in bleached synapses may correspond to the Proline Rich Membrane Anchor protein (PRiMA)/AChE population. Indeed, it has been shown that PRiMA can organize AChE tetramers and anchor them in plasma membranes (Perrier et al., 2002), although the proportion of AChE anchored by PRiMA in the plasma membrane remains unsolved. In any event, it would be interesting to study the mobility of AChE in mice deficient in PRiMA.
In contrast to nonsynaptic AChE mobility, our data argue that synaptic AChE is almost entirely immobile or that it has a very slow diffusion within the synapse, at least in the time period of our analyses. Whether manipulation of synaptic activity would alter the mobility of this AChE population remains to be seen. AChE gene expression has been shown to be sensitive to electrical activity (Michel et al., 1994). Our data indicate that the majority of AChE at synapses is directly inserted into synaptic cleft (Figure 1E). These results are supported by previous reports showing the presence of high local expression of both AChE and ColQ mRNAs in fast muscles (Jasmin et al., 1993; Legay et al., 1995; Krejci et al., 1999). However, the lack of the mobility of synaptic AChE may indicate that this population of AChE is tethered very tightly in synaptic basal lamina through the scaffold ColQ-perlecan-dystroglycan. Interestingly, in mice deficient in perlecan and dystroglycan, AChE is undetectable (Jacobson et al., 2001; Arikawa-Hirasawa et al., 2002); and more recently, it has been shown that AChE anchoring in heterologous cell systems requires muscle-specific kinase through its interaction with the C terminus of ColQ (Cartaud et al., 2004).
At present, it is not clear how intracellular alpha dystrobrevin protein could affect extracellular matrix AChE stability, because it does not link directly to AChEs. A reduction or absence of AChE clusters has been reported in the synapses of several mouse mutants. For example in mice deficient in perlecan, AChE is totally lacking at neuromuscular synapses (Arikawa-Hirasawa et al., 2002), and AChE is significantly reduced or no longer concentrated at dystroglycan null synapses (Jacobson et al., 2001). Similarly, in mice deficient in the protein rapsyn, which is associated with AChRs, AChRs do not cluster and AChE aggregates also fail to form at synaptic sites, suggesting that rapsyn–AChR interaction is also essential for aggregation of AChE at the basal lamina. In the present work, we report that the density and turnover rate of AChEs are dramatically affected in adbn–/– synapses (Figures 3 and 4). Because most removed AChEs were replaced by newly synthesized ones, our findings suggest that adbn may be involved in AChE stability rather than synthesis. As with AChE, we previously showed that the density and turnover rate of AChRs are also significantly affected in adbn–/– synapses (Grady et al., 2000; Akaaboune et al., 2002). These findings, along with our previous results on AChR dynamics, clearly show that there is a correlation between number and lifetime of AChEs and the number and lifetime of AChRs at individual synapses. Although it is not clear that a direct interaction exists between AChR and AChE, it is possible that the loss of -dystrobrevin primarily affects the number of AChRs through changes in conformation of the DGC and/or its association with other postsynaptic components; changes in AChRs in turn may affect the number and stability of AChE. This idea is supported by the following evidence: 1) there are no AChE clusters when AChR clusters do not form (De La Porte et al., 1998); 2) when the number of AChRs decreases, the number of AChEs decreases by the same magnitude; and 3) when the turnover of AChR increases the AChE turnover rate also increases. Clustering of AChR could be the starting point for accumulation of AChE, although there is no evidence to date for a direct interaction between AChR and AChE. Nevertheless, it has been shown that the loss of AChE can control the density of AChRs. For example, in mutants lacking ColQ (in which AChE fails to concentrate at the synapse) or in AChE–/– mutants, receptor density is significantly decreased (Feng et al., 1999; Xie et al., 2000). It would be interesting to determine whether AChE lifetime is affected in these mice. An alternative idea to account for adbn effects on AChE is that the high turnover rate and low number of AChE results from a reduction of dystroglycan, which provides a molecular link between the basal lamina and intracellular scaffold proteins. However, previous studies and our data argue against this possibility, because neither perlecan nor dystroglycan seem to be affected by the loss of adbn–/– (Grady et al., 1999). It is possible that the loss of -dystrobrevin may indirectly reduce the level of other DGC components that, in turn, interact with other extracellular synaptic components, thereby altering the stability of AChE. For example, a similar phenotype was observed in mutant mice lacking -syntrophin, another component of the DGC, in which AChRs and AChEs are decreased at the NMJ (Adams et al., 2000).
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ACKNOWLEDGMENTS |
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-dystrobrevin mutant mice. This work was supported by the University of Michigan, National Institute of Neurological Disorders and Stroke Research grant NS-047332 (to M.A.). |
Footnotes |
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Address correspondence to: Mohammed Akaaboune (makaabou{at}umich.edu).
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