A Novel Mechanism for Modulating Synaptic Gene Expression: Differential Localization of α-Dystrobrevin Transcripts in Skeletal Muscle

Molecular and Cellular Neuroscience 17, 127–140 (2001) doi:10.1006/mcne.2000.0918, available online at http://www.idealibrary.com on

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A Novel Mechanism for Modulating Synaptic Gene Expression: Differential Localization of ␣-Dystrobrevin Transcripts in Skeletal Muscle Sarah E. Newey,* Anthony O. Gramolini, † Jun Wu, † Paul Holzfeind,* ,1 Bernard J. Jasmin, † Kay E. Davies, ‡,2 and Derek J. Blake* *Department of Human Anatomy and Genetics, ‡MRC Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford, OX1 3QX, United Kingdom; and †Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, Ontario, K1H 8M5, Canada

␣-Dystrobrevin is a dystrophin-related and -associated protein that is involved in synapse maturation and is required for normal muscle function. There are three protein isoforms in skeletal muscle, ␣-dystrobrevin-1, -2, and -3 that are encoded by the single ␣-dystrobrevin gene. To understand the role of these proteins in muscle we have investigated the localisation and transcript distribution of the different ␣-dystrobrevin isoforms. ␣-Dystrobrevin-1 and -2 are concentrated at the neuromuscular junction and are both recruited into agrin-induced acetylcholine receptor clusters in cultured myotubes. We also demonstrate that all ␣-dystrobrevin mRNAs are transcribed from a single promoter in skeletal muscle. However, only transcripts encoding ␣-dystrobrevin-1 are preferentially accumulated at postsynaptic sites. These data suggest that the synaptic accumulation of ␣-dystrobrevin-1 mRNA occurs posttranscriptionally, identifying a novel mechanism for synaptic gene expression. Taken together, these results indicate that different isoforms possess distinct roles in synapse formation and possibly in the pathogenesis of muscular dystrophy.

INTRODUCTION An important event in the formation of the neuromuscular junction (NMJ) is the compartmentalisation of postsynaptic components opposite the nerve terminal.

1 Present address: Neuromuskulaere Abteilung, Anatomie III, Waehringerstrasse 13, A-1090, Vienna, Austria. 2 To whom correspondence and reprint requests should be addressed. Fax: ⫹1865 272420. E-mail: [email protected]

1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Two proteins, agrin and neuregulin (ARIA, acetylcholine receptor inducing activity), are secreted from the nerve terminal to orchestrate this process. Agrin triggers the posttranslational clustering of acetylcholine receptors (AChRs) along with other postsynaptic proteins including the dystrophin-related protein utrophin (reviewed by Ruegg and Bixby, 1998). Neuregulin modulates the synapse-specific transcription of several postsynaptic proteins including AChRs and utrophin (reviewed by Sanes and Lichtman, 1999). Its effect is mediated by the transcription factor GABP (GA binding protein), which binds to the N-box element in the promoter regions of AChR subunit and utrophin genes (Schaeffer et al., 1998; Fromm et al., 1998; Dennis et al., 1996; Gramolini et al., 1999; Khurana et al., 1999). Proteins of the dystrophin-associated protein complex (DPC) are also implicated in the formation and maintenance of the NMJ. The dystrophin-deficient mdx mouse and utrophin-deficient mice have abnormalities in the postsynaptic membrane (Lyons and Slater, 1991; Deconinck et al., 1997a; Grady et al., 1997a). These findings have led to the proposal that members of the dystrophin family form a specific scaffold at the NMJ (Jasmin et al., 1990; Xu and Salpeter, 1997; reviewed by Sanes et al., 1998). Furthermore, the dystrophin-associated protein ␣-dystroglycan is the major agrin-binding protein in muscle, although this interaction does not appear to mediate the AChR clustering effect of agrin (Gesemann et al., 1996). ␤-dystroglycan, on the other hand, interacts with rapsyn, a cytoplasmic protein absolutely required for the agrin-induced clustering of AChRs (Apel et al., 1995; Cartaud et al., 1998).

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128 Thus, the postsynaptic cytoskeleton of the NMJ contains a specialised DPC that is required to maintain normal synaptic structure. ␣-Dystrobrevin mutant mice provide the most compelling evidence for a role for the DPC at peripheral synapses. These mice have altered synaptic morphology and destabilised AChR clusters (Grady et al., 2000). ␣-Dystrobrevin is a dystrophin-related protein that binds directly to dystrophin and utrophin via reciprocal coiled-coil domains (Sadoulet-Puccio et al., 1997). This protein was originally identified as an 87-kDa phosphoprotein in the Torpedo electric organ that copurified with the AChRs, rapsyn, syntrophin, and dystrophin (Carr et al., 1989; Butler et al., 1992; Wagner et al., 1993; Kramarcy et al., 1994; Dwyer and Froehner et al., 1995). In mammalian muscle there are at least three isoforms: ␣-dystrobrevin-1 (94 kDa), ␣-dystrobrevin-2 (62 kDa), and ␣-dystrobrevin-3 (42 kDa), which are generated by alternative splicing of the single gene (Blake et al., 1996; Ambrose et al., 1997; Sadoulet-Puccio et al., 1996; Nawrotzki et al., 1998; Enigk and Maimone, 1999). While ␣-dystrobrevin-1 and -2 contain dystrophin and syntrophin binding sites, ␣-dystrobrevin-3 lacks both of these sites (Nawrotzki et al., 1998; Sadoulet-Puccio et al., 1997). In addition to synaptic defects, ␣-dystrobrevin mutant mice have muscular dystrophy, indicating that these proteins play pleiotropic roles in muscle (Grady et al., 1999). However, the relative contributions of each isoform to these processes is unknown. In view of these findings, we have investigated the localisation and regulation of the different ␣-dystrobrevin isoforms at the postsynaptic membrane and their involvement in muscular dystrophy. We have shown that although the transcripts for ␣-dystrobrevins-1, -2, and -3 originate from the same promoter in skeletal muscle, they differentially accumulate at the NMJ. The selective synaptic accumulation of ␣-dystrobrevin-1 transcripts is likely to occur posttranscriptionally and thereby represents a novel mechanism for synaptic gene expression. We also show that there is preferential loss of ␣-dystrobrevin-3 protein in dystrophin-deficient muscle. These results suggest that the three dystrobrevin isoforms contribute differently to the maintenance of the postsynaptic membrane and assembly of the DPC at the sarcolemma.

RESULTS ␣-Dystrobrevin Isoforms in Skeletal Muscle Previously we have described multiple ␣-dystrobrevin transcripts that encode three protein isoforms,

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␣-dystrobrevin-1, -2, and -3, in mouse skeletal muscle (Blake et al., 1996; Ambrose et al., 1997; Nawrotzki et al., 1998) (Fig. 1). In order to localize these ␣-dystrobrevin isoforms in skeletal muscle, we generated a panel of specific antibodies and used them to immunolabel normal and dystrophin-deficient mdx mouse quadricep cryosections. Using the antibody ␣1CT-FP (Blake et al., 1998), ␣-dystrobrevin-1 immunoreactivity was concentrated at the NMJ of normal mouse muscle, with much weaker labeling detected at the sarcolemma (Figs. 2A and 2B). In mdx muscle, the sarcolemmal labeling is largely absent, although strong labeling of the NMJs remains (Figs. 2C and 2D). The peptide antibody ␣2PEP was generated against the unique C-terminus of ␣-dystrobrevin-2 and in agreement with previous work (Peters et al., 1998) demonstrated strong labelling of the sarcolemma and NMJ (Figs. 2E and 2F). This staining was completely abolished by preincubation of ␣2-PEP with the immunising peptide (data not shown). The sarcolemmal labeling of ␣2-PEP is dramatically reduced in mdx muscle, suggesting that association with dystrophin is the major factor in localising ␣-dystrobrevin-2 to the muscle membrane (Figs. 2G and 2H). In contrast, the NMJs of mdx muscle remain strongly labeled with ␣2-PEP. Immunolabeling with the antibody ␣-PAN, designed to detect all three ␣-dystrobrevin isoforms, gave strong labeling at the sarcolemma and NMJ (Figs. 2I and 2J). This result implies that ␣-dystrobrevin-3 is also likely to be localized to the sarcolemma and NMJ since no additional labeling away from the membrane is detected. As expected, the sarcolemmal labeling was dramatically reduced in mdx muscle while NMJs were still strongly stained (Figs. 2K and 2L). Characterization of this antibody demonstrated that ␣-PAN detects all three ␣-dystrobrevin isoforms in COS-7 cells transfected with ␣-dystrobrevin expression constructs and that staining can be completely abolished by peptide blocking experiments (data not shown). Western blots were used to demonstrate that ␣-dystrobrevin-3 can be detected in muscle and to provide evidence that, like ␣-dystrobrevins-1 and -2 (SadouletPuccio et al., 1997; Nawrotzki et al., 1998; Peters et al., 1998), this isoform is likely to be associated with dystrophin at the sarcolemma. Western blots of total protein extracted from normal and mdx tissues were probed with the antibody ␣-PAN (Fig. 3). All three ␣-dystrobrevin isoforms can be detected with this antibody on western blots, but it does not cross react with the related protein ␤-dystrobrevin (Peters et al., 1997; Blake et al., 1998). This experiment shows that ␣-dystrobrevin-3, which has a predicted molecular mass of 42 kDa (Nawrotzki et al., 1998), is severely reduced in mdx

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FIG. 1. ␣-Dystrobrevin Isoforms in Skeletal Muscle and their 3⬘ untranslated regions (UTRs). Schematic diagram of the ␣-dystrobrevin transcripts found in skeletal muscle (Ambrose et al., 1997). Wide unshaded boxes represent the coding region with identifiable domains marked: EF, EF hand region; ZZ, ZZ domain (Ponting et al., 1996); CC, coiled-coil domain (Blake et al., 1995); Y, unique tyrosine kinase substrate domain of ␣-dystrobrevin-1. Thin boxes represent the unique 3⬘UTRs of each isoform: black boxes indicate the two different 3⬘UTRs identified for ␣-dystrobrevin-1; white boxes indicate the two different 3⬘UTRs for ␣-dystrobrevin-2 and the vertically striped box represents the 3⬘UTR for ␣-dystrobrevin-3. Angled lines indicate splicing to the internal acceptor site in the UTR exon of ␣-dystrobrevin-1. The two alternative ␣-dystrobrevin-2 3⬘UTR sequences are generated by differential usage of polyadenylation signal sequences (Ambrose et al., 1997). The positions of the isoform specific antisense oligonucleotide probes used in subsequent in situ hybridization experiments are indicated: DB1, ␣-dystrobrevin-1 probe; DB2, ␣-dystrobrevin-2 probe; and DB3, ␣-dystrobrevin-3 probe.

muscle and heart. The presence of two bands at approximately 40 kDa may reflect posttranslational modification or alternative splicing of ␣-dystrobrevin-3. The severe reduction of ␣-dystrobrevin-3 in mdx muscle suggests that the DPC is required for the maintenance of this protein. Levels of ␣-dystrobrevin-1 and -2 are also reduced in mdx skeletal muscle, while ␣-dystrobrevin-2 is reduced in mdx heart. In contrast, levels of ␣-dystrobrevin-1 and -2 are unaffected in mdx brain (Fig. 3).

␣-Dystrobrevin-1 and ␣-Dystrobrevin-2 Are Recruited into Agrin-Induced AChR Clusters in C2C12 Myotubes In order to investigate the involvement of ␣-dystrobrevins in synapse formation, we examined the localization and clustering of ␣-dystrobrevin isoforms in

C2C12 myotubes. We have previously shown that ␣-dystrobrevin-1 is recruited into agrin-induced AChR macroclusters in C2C12 myotubes along with utrophin (Nawrotzki et al., 1998). To investigate whether ␣-dystrobrevin-2 is also aggregated into agrin-induced macroclusters, day 6 C2C12 myotubes were treated with a clustering proficient agrin isoform C95 A4B8 (Gesemann et al., 1995). Untreated cultures were used as a control. Myotubes were processed for immunofluorescence and double labeled with ␣-bungarotoxin and the antibody ␣2-PEP (Figs. 4C and 4D). ␣-Dystrobrevin-2 labeling was detected throughout the myotubes but was clearly concentrated in clusters that colocalized with both spontaneous and agrin-induced AChR clusters in a manner similar to ␣-dystrobrevin-1 (Figs. 4A and 4B). Quantification of the clustering revealed that agrin treatment of C2C12s induced between four- and fivefold induction of AChR macroclusters with the conco-

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FIG. 2. Localization of ␣-dystrobrevin isoforms in skeletal muscle. Normal C57 and mdx mouse skeletal muscle cryosections were double labeled with Alexa-488-conjugated ␣-bungarotoxin (B, D, F, H, J, L, N, P) and antibodies ␣1CT-FP, which is specific for ␣-dystrobrevin-1 (A, C), ␣2-PEP, which is specific for ␣-dystrobrevin-2 (E, G), ␣-PAN, which recognizes all three ␣-dystrobrevin isoforms (I, K) and without primary antibody (M, O). ␣-Dystrobrevin-1 and -2 are located at the sarcolemma and NMJ of normal muscle. By elimination, staining with the ␣-PAN antibody implies that ␣-dystrobrevin-3 is also located to the sarcolemma since there are no obvious differences in immunostaining. In mdx muscle, the sarcolemmal labeling of ␣-dystrobrevin-1 and -2 is dramatically reduced although strong junctional staining is retained. Scale bar, 50 ␮m.

mittant recruitment of both ␣-dystrobrevin-1 and -2 to these clusters (Figs. 4E and 4F). Synaptic Accumulation of ␣-Dystrobrevin Transcripts in Skeletal Muscle To determine the localisation of the different ␣-dystrobrevin transcripts in skeletal muscle, in situ hybridizations were performed using isoform-specific antisense oligonucleotide probes (Fig. 1). Our results showed a selective accumulation of ␣-dystrobrevin-1

transcripts within the postsynaptic sarcoplasm of muscle fibers (Figs. 5A–5D) in a pattern similar to that described for utrophin mRNAs (Gramolini et al., 1997; Vater et al., 1998). NMJs were initially scored according to whether they showed transcript accumulation. This analysis revealed that ␣-dystrobrevin-1 transcripts were synaptically accumulated at over 70% of NMJs examined (Fig. 5I). mRNAs corresponding to ␣-dystrobrevin-1 were also detected in extrasynaptic regions of muscle fibers but densitometric analysis demonstrated that levels of ␣-dystrobrevin-1 mRNA confined within

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131 the possibility of technical failure (Fig. 5J). These results demonstrate a clear difference between the localization of ␣-dystrobrevin-1 transcripts and mRNAs corresponding to ␣-dystrobrevins-2 and -3 in skeletal muscle.

All Three ␣-Dystrobrevin Isoforms Are Transcribed from a Single Promoter in Skeletal Muscle

FIG. 3. ␣-Dystrobrevin isoforms are reduced in mdx skeletal muscle and heart. 20 ␮g of total protein from normal C57 and mdx heart, skeletal muscle, and brain was separated on an 8% SDS–PAGE gel, western blotted, and probed with the antibody ␣-PAN, which recognizes all three ␣-dystrobrevin protein isoforms. An identical gel was stained with Coomassie brilliant blue to confirm loading of all lanes was equal (data not shown). In skeletal muscle and heart ␣-dystrobrevin-1 and -2 contain the muscle expressed vr3 sequence (Blake et al., 1996), which accounts for the larger proteins detected in these tissues when compared to brain. Note the reduction in the total amount of all three ␣-dystrobrevin isoforms in mdx skeletal muscle and the reduction of ␣-dystrobrevin-2 and -3 in mdx heart. However, levels of ␣-dystrobrevin-1 and -2 are unaffected in mdx brain. Molecular mass markers (in kDa) are indicated on the right.

the postsynaptic sarcoplasm were approximately fivefold higher than those observed in extrasynaptic regions (Fig. 5J). In control experiments performed with sense oligonucleotides, these probes failed to label subcellular structures above background levels (Figs. 5E, 5F, and 5J). Using antisense probes that specifically detect ␣-dystrobrevin-2 and ␣-dystrobrevin-3 mRNAs, a different pattern was observed. Both ␣-dystrobrevin-2 and -3 transcripts were found to be largely distributed throughout the muscle sarcoplasm and were found to be accumulated at only 28% of NMJs examined (Figs. 5G–5I). Furthermore, using densitometric analysis, the extent of enrichment at the postsynaptic sarcoplasm was only 1.5-fold greater than in the extrasynaptic sarcoplasm (Fig. 5J). Again, sense oligonucleotides were used as control probes and these failed to label the muscle fibers. Both antisense oligonucleotides resulted in extrasynaptic signals that were approximately 10fold greater than the sense control probes, ruling out

In order to understand how ␣-dystrobrevin-1 transcripts are accumulated at synapses we examined the promoter usage of the different ␣-dystrobrevins isoforms in skeletal muscle. Previously we have shown that ␣-dystrobrevin is transcribed from three different promoters, A, B, and C, which appear to be active in a tissue selective manner (Holzfeind et al., 1999). In skeletal muscle, the synapse-specific transcription of ␣-dystrobrevin-1 may be regulated by an isoform-specific promoter employing an N-box, as described for AChRs (Schaeffer et al., 1998), utrophin (Dennis et al., 1996; Gramolini et al., 1999; Khurana et al., 1999) and acetylcholinesterase (AChE) (Chan et al., 1999). Alternatively, all ␣-dystrobrevin transcripts maybe transcribed from a single promoter in skeletal muscle, thus identifying a posttranscriptional mechanism for the targeting of ␣-dystrobrevin-1 transcripts to the NMJ. To determine the promoter utilisation of the ␣-dystrobrevin isoforms in muscle, a series of RT-PCR reactions were performed to amplify transcripts encoding each of the ␣-dystrobrevin isoforms originating from the three different promoters A, B, and C (Fig. 6). Isoform specific primers were designed to the unique Cterminal-encoding regions of each ␣-dystrobrevin isoform. These primers were used with three forward primers located in the 5⬘ UTRs downstream of promoters A, B, and C (Fig. 6). This combination of primers allowed the detection of ␣-dystrobrevin-1, -2, and -3 transcripts originating from the three promoters in skeletal muscle and brain. To aid interpretation of these results, PCR products were dot blotted onto Hybond N⫹ membrane in an arrangement shown in Fig. 6 and probed with a radiolabeled ␣-dystrobrevin probe. Southern blot analysis of the PCR products revealed single bands of the expected size in brain and skeletal muscle (data not shown). These experiments show that all ␣-dystrobrevin transcripts expressed in skeletal muscle originate from a single promoter, promoter C. In contrast, control experiments in brain demonstrate that all three promoters transcribe the different ␣-dystrobrevin isoforms.

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FIG. 4. Agrin-induced clustering of ␣-dystrobrevin-1 and -2 in C2C12 myotubes. Day 6 C2C12 myotubes were treated with or without 5 nM C95A4B8 agrin for 16 h. Parallel cultures were fixed and permeabilized and double-labeled with Alexa-488 ␣-bungarotoxin (B, D) and either ␣1CT-FP (A) or ␣2-PEP (C). Note the colocalization of ␣-dystrobrevin-1 and -2 with AChR.* Scale bar, 50 ␮m. (E and F) Quantification of agrin induced clustering of AChRs, ␣-dystrobrevins-1 and ␣-dystrobrevin-2. Myotubes were treated as described above. For control and agrin stimulated cultures, 18 random fields of view were photographed twice: first for ␣-bungarotoxin fluorescence and second for the ␣-dystrobrevin isoform fluorescence. The total number of AChR and ␣-dystrobrevin-1 (E) or ␣-dystrobrevin-2 (F) macroclusters were counted and the mean (⫾SEM) calculated. Treatment with agrin induced an approximately threefold increase in ␣-dystrobrevin-1-positive macroclusters and an approximately fourfold increase of ␣-dystrobrevin-2 positive macroclusters. In all conditions, addition of agrin produced a highly significant increase (*) in the number of macroclusters (P ⬍ 0.001, t test). *Clusters.

DISCUSSION Recent work characterising ␣-dystrobrevin-deficient mice demonstrates that while the ␣-dystrobrevins are dispensable for the formation of NMJ, they are absolutely required for the maturation of the postsynaptic apparatus (Grady et al., 2000). In this paper we investi-

gate the synaptic regulation of the different ␣-dystrobrevin isoforms in skeletal muscle. In agreement with previous work, ␣-dystrobrevin-1 and -2 are concentrated at the NMJ (Peters et al., 1998). We also show that both isoforms are localized to the sarcolemma where ␣-dystrobrevin-2 is more abundant than ␣-dystrobrevin-1. In the absence of dystrophin, this sarcolemmal

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localization of ␣-dystrobrevin-1 and -2 is largely lost, demonstrating the importance of dystrophin and the DPC in localizing these isoforms to the muscle membrane. At the NMJ in mdx muscle, both isoforms remain localized to the postsynaptic membrane, suggesting that the mechanism for anchoring these complexes at the junction is different from that at the sarcolemma. This observation may be explained by an association between ␣-dystrobrevin and utrophin at the NMJ. However, in utrophin-deficient muscle and in muscle lacking both dystrophin and utrophin, ␣-dystrobrevin-1 and -2 remain localized at the NMJ (Newey and Blake, unpublished observations; Peters et al., 1998) along with other components of the DPC (Deconinck et al., 1997b; Grady et al., 1997b). This implies that other, unidentified proteins are responsible for localizing DPC components to the NMJ. Both ␣-dystrobrevin-1 and -2 are recruited into agrininduced AChR clusters in cultured myotubes. This observation indicates that both isoforms could contribute to the stabilization of these specialisations. It has been suggested that ␣-dystrobrevin-2 is probably not an important mediator of synaptic stability and that ␣-dystrobrevin-1 is more likely to fulfil this function (Grady et al., 2000). However, our results support the conclusion that ␣-dystrobrevin-2 may indeed have a role at the synapse. In support of this idea, Peters et al. (1998) finds that ␣-dystrobrevin-2 is colocalized with sodium channels in the troughs of the junctional folds. Sodium channels are also clustered by agrin (Sharp and Caldwell, 1996), which leads to the prediction that while ␣-dystrobrevin-1 may be important for the stabilization of AChRs at the crests of the postsynaptic folds, ␣-dystrobrevin-2 may be required for the correct maintenance of sodium channels at the base of these folds. Our data also demonstrate that ␣-dystrobrevin-3 is a component of both skeletal muscle and heart, and that levels of this protein are severely reduced in mdx tissue. This result suggests that, like ␣-dystrobrevins-1 and -2, ␣-dystrobrevin-3 is likely to be associated with dystrophin at the sarcolemma (Sadoulet-Puccio et al., 1997; Nawrotzki et al., 1998; Peters et al., 1998). ␣-Dystrobrevin-3 was thought unlikely to be part of the DPC since this isoform lacks the coiled-coil domain shown to mediate the direct interaction of ␣-dystrobrevin-1 and -2 with dystrophin (Sadoulet-Puccio et al., 1997) and also lacks the proposed syntrophin binding site (Dwyer and Froehner, 1995). Our results indicate that the N-terminus of the ␣-dystrobrevins may be important in mediating their associations with the DPC and imply that ␣-dystrobrevin-3 contributes to maintaining normal muscle stability. This observation is supported by re-

133 cent results from which indicate that ␣-dystrobrevin-3 is associated with the sarcoglycan complex in skeletal muscle (Yoshida et al., 2000). While ␣-dystrobrevin-1 and -2 are clearly components of the postsynaptic membrane of the NMJ (Peters et al., 1998), we show that the transcripts encoding these proteins are differentially localized in skeletal muscle. ␣-Dystrobrevin-1 transcripts are highly accumulated underneath the majority of synapses examined, whereas mRNAs for ␣-dystrobrevin-2 and -3 demonstrate only a modest accumulation at NMJs and are more evenly distributed throughout the muscle sarcoplasm. This modest accumulation of ␣-dystrobrevin-2 and -3 transcripts maybe attributable to the accumulation of myonuclei underneath the NMJ. As a result, any transcript that is synthesized at the same rate by myonuclei might be expected to have limited accumulation at the NMJ. These in situ results not only precisely reflect the localisation of the corresponding proteins in normal muscle, but are particularly important because we find that all three ␣-dystrobrevin isoforms are transcribed from a single promoter (promoter C) in skeletal muscle. Taken together, these data imply that the synaptic accumulation of ␣-dystrobrevin-1 transcripts occurs posttranscriptionally and proposes a novel mechanism for the localization of synaptic proteins. Transcription of other postsynaptically regulated genes, the AChR subunits, utrophin and AChE, is controlled by a neuregulin-dependent pathway whose downstream targets act upon N-box sequences in these genes. In the case of AChRs and utrophin, the N-box is located within the promoters (Koike and Changeux, 1995; Dennis et al., 1996), whereas the essential N-box is found within the first intron of the AChE gene (Chan et al., 1999). Analysis of the ␣-dystrobrevin promoter C did not reveal an N-box element even though multiple sequences known to mediate skeletal muscle expression were found (Holzfeind et al., 1999). Furthermore, the influence of an N-box, even remote from the promoter, is unlikely to affect the selective synaptic accumulation of ␣-dystrobrevin-1 transcripts since all three ␣-dystrobrevin isoforms contain identical first coding exons transcribed from the same promoter (Blake et al., 1996; Ambrose et al., 1997; Holzfeind et al., 1999). However, it is also possible that enhancer sequences, possibly remote from the promoter, may be responsive to local transcription factors to modulate the production of the different ␣-dystrobrevin isoforms in different regions of the muscle fiber. One possible explanation for our results is that a fourth, as yet unidentified, promoter specifically transcribes a synaptically accumulated ␣-dystrobrevin-1

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FIG. 5. Detection of ␣-dystrobrevin transcripts in skeletal muscle by in situ hybridization. (A, C, and E) Representative bright field photomicrograph of a skeletal muscle cryosection stained for AChE to visualize neuromuscular junctions. (B and D) Corresponding serial sections processed for in situ hybridization using ␣-dystrobrevin-1-specific antisense probe. Comparison of these panels reveals the accumulation of ␣-dystrobrevin-1 mRNAs within the postsynaptic sarcoplasm. Scale bar, 45 ␮m. (F) Corresponding in situ hybridization using sense ␣-dystrobrevin-1 probe demonstrating the specificity of the labeling. Scale bar, 90 ␮m. (G and H) Localization of ␣-dystrobrevin-2 mRNAs in

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FIG. 6. All ␣-dystrobrevin transcripts are transcribed from promoter C in skeletal muscle. The top panel shows a schematic representation of the organization of the 5⬘-end of the ␣-dystrobrevin gene (Holzfeind et al., 1999). The three promoters (A–C) are shown associated with their downstream 5⬘UTR exons. The first coding exon and subsequent exons where isoform specific reverse primers were generated are also shown. RT PCRs were performed on first strands prepared from skeletal muscle and brain using UTR-specific forward primers (UTRAf, UTRBf, and UTRCf) and isoform-specific reverse primers (DB1r, DB2r, and DB3r). Negative control RT PCRs were set up without template. Using these combinations of primers, amplification of ␣-dystrobrevin-1, -2, and -3 transcripts originating from each of the three promoters can be achieved. The lower panel shows the resulting dot blot of the PCR reactions. While it was possible to amplify transcripts for all three ␣-dystrobrevin isoforms containing each of the 5⬘ UTR exons A–C in brain, in skeletal muscle only exon C containing transcripts could be amplified which encode all three isoforms.

skeletal muscle. (G) Representative bright field muscle section processed for AChE visualization to identify NMJs. (H) Corresponding serial section processed for in situ hybridization of ␣-dystrobrevin-2. Note the small amount of accumulation of ␣-dystrobrevin-2 mRNAs at the NMJ indicated with an arrowhead. (I) Quantification of the accumulation of ␣-dystrobrevin-1, -2, and -3 mRNAs at NMJs. Analysis of ␣-dystrobrevin-1 in situ hybridizations revealed that of the 119 NMJs examined, 84 (approx 71%) displayed an accumulation of silver grains corresponding to ␣-dystrobrevin-1 transcripts. In contrast, only 28% of NMJs showed an accumulation of either ␣-dystrobrevin-2 mRNAs (55/191) or ␣-dystrobrevin-3 mRNAs (48/170). Results for the ␣-dystrobrevin-1 sense control probe are shown. Similar results were obtained with ␣-dystrobrevin-2 and -3 sense probes. (J) Quantification of the levels of ␣-dystrobrevin-1, -2, and -3 mRNAs in synaptic verses extrasynaptic regions of skeletal muscle fibers. The labeling density in synaptic verses extrasynaptic regions was determined by measuring the number of labeled pixels within junctional and extrajunctional regions of the muscle. Note that all antisense probes resulted in similar levels of extrasynaptic labeling, approximately 10-fold greater than the sense controls. A total of 50 measurements were taken for each in situ probe.

136 transcript in skeletal muscle. However, this is unlikely since we have shown by northern blotting that ␣-dystrobrevin-1 transcripts originating from promoter C are detected at similar levels to those detected with a common ␣-dystrobrevin probe (Holzfeind et al., 1999). Furthermore, in situ hybridizations on skeletal muscle using an antisense oligonucleotide probe specific for the 5⬘UTR exon downstream of promoter C revealed synaptic accumulation of transcripts at approximately 43.5% (112/257) NMJs examined, implying that a proportion of mRNAs originating from promoter C are concentrated underneath the synapse (data not shown). Finally, the organization of the ␣-dystrobrevin promoters is very similar to that described for the related dystrophin locus, where three tissue selective promoters regulate the expression of full-length dystrophin isoforms (Nudel et al., 1989; Gorecki et al., 1992). Taken together, we have no evidence to support the existence of a fourth ␣-dystrobrevin-1-specific promoter. Hence, we propose that an alternative, promoterindependent mechanism is responsible for the synaptic localization of ␣-dystrobrevin-1 transcripts in skeletal muscle. The simplest mechanism that can be proposed to explain these observations involves the specific stabilization of ␣-dystrobrevin-1 mRNAs at the synapse. Indeed, several studies have shown that transcript stabilization plays a significant role in the regulation of AChE in differentiating myogenic, neuronal, and hematopoietic cells maintained in culture (Fuentes and Taylor, 1993; Coleman and Taylor, 1996; and Chan et al., 1998) as well as in denervated muscles, most likely via alterations in the pattern of RNA-protein interactions (Boudreau-Larivie`re et al., 2000). An alternative, but more complex, explanation for these observations may involve the specific direction of ␣-dystrobrevin-1 transcripts to the synapse. Transcript localization systems have been described in both differentiating and differentiated cells and involve targeting regions, or “zipcodes,” which are found in the 3⬘UTRs of the mRNAs (reviewed by Oleynikov and Singer, 1998). These sequences interact with specific RNA binding proteins that in turn mediate interactions with cytoskeletal elements to direct transcripts to the relevant site. Unfortunately, in most cases no consensus sequences exist for the zip codes, making the identification of such sequences difficult (Oleynikov and Singer, 1998). Recently, a novel mechanism has been described that targets different mRNA products of a single gene to distinct intracellular destinations within the Drosophila ovary (Whittaker et al., 1999). In this system, four classes of mRNAs are produced from the hu-li tai shao (hts)

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gene, each of which encodes a distinct protein isoform. Each of the four hts mRNAs possess a unique 3⬘UTRs that either do, or do not, contain transport and localization elements (Whittaker et al., 1999). Similarly, each of the ␣-dystrobrevin transcripts are assigned a unique 3⬘UTR sequence (Ambrose et al., 1997). It is possible that these 3⬘UTR sequences contain the spatial information necessary to direct the ␣-dystrobrevin mRNAs to the appropriate subcellular location. In the case of ␣-dystrobrevin-1, it is possible that the 3⬘UTR is responsible for directing the synapse-specific accumulation of mRNAs. While the precise molecular mechanisms involved in the stabilisation or direction of ␣-dystrobrevin-1 transcripts to the NMJ remain to be confirmed, the importance of localized postsynaptic translation in shaping synapses is beginning to be understood. Recent work has shown that postsynaptic translation affects the efficacy and morphology of neuromuscular junctions in Drosophila (Sigrist et al., 2000). Thus, the mechanisms employed to direct and translate synaptic components are likely to influence the development, function and organisation of synapses.

EXPERIMENTAL METHODS Antibodies The polyclonal antibody ␣1CT-FP specifically detects ␣-dystrobrevin-1 and is raised against the unique Cterminus of ␣-dystrobrevin-1 (Blake et al., 1998). ␣1CTFP antiserum was affinity purified using the immunising fusion protein coupled to Sulfolink Coupling Gel (Pierce) according to the manufacturer’s instructions. In addition, a polyclonal antibody that is specific for ␣-dystrobrevin-2 (␣2-PEP) and an antibody that detects all three dystrobrevin isoforms (␣-PAN) were prepared by immunising rabbits with synthetic peptides. Antibody ␣2-PEP was generated against the peptide NH 2KQGVSYVPYCRS, corresponding to the 12 carboxyterminal amino acids of ␣-dystrobrevin-2. Antiserum was affinity purified using the peptide coupled to NHSactivated Sepharose (Amersham Pharmacia Biotech) according to manufacturer’s instructions. The ␣-PAN antibody was generated against the peptide NH 2GSPFITRSSDGAHGGC and affinity purified with peptide coupled to SulfoLink Coupling Gel (Pierce). All antibodies were eluted using ImmunoPure IgG elution buffer (Pierce).

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Cell Culture and Agrin Treatment C2C12 mouse muscle cells (Yaffe and Saxel, 1977) were routinely cultured in DMEM supplemented with 10% fetal calf serum (PAA Laboratories), 2 mM glutamine, and penicillin/streptomycin. For immunofluorescence studies, cells were plated onto two-well chamber slide flasks (Labtek) and stained as described above. Myoblast fusion was induced by culturing the cells in DMEM supplemented with 5% horse serum, glutamine, and antibiotics (differentiation medium). The production of recombinant agrin is described elsewhere (Gesemann et al., 1995). Day 6 differentiated myotubes were treated with 5 nM agrin diluted in fresh differentiation medium for 16 h at 37°C prior to fixation. Control myotubes were treated with fresh differentiation medium without agrin. Immunofluorescence Microscopy Eight-micrometer cryosections were prepared from adult quadriceps of normal C57 and dystrophin-deficient mdx mice. Sections were incubated for 30 min in phosphate-buffered saline (PBS) containing 10% fetal calf serum followed by a 1 h incubation with primary antibody, diluted in PBS. Antibody dilutions were 1:100 for ␣2-pep, 1:200 for ␣-pan, and 1:1000 for ␣1CT-FP polyclonal antiserum. Sections were washed twice in PBS and incubated for 1 h with rhodamine-red-X-conjugated donkey anti-rabbit IgG, diluted 1:100 in PBS (Jackson Immunoreseach Laboratories, Inc.) and Alexa 488-conjugated ␣-bungarotoxin, diluted 1:500 (Molecular Probes, Inc.). Sections were washed twice in PBS and mounted in Vectashield mounting medium (Vector Laboratories). Labeled sections were analyzed and photographed using a Leica DM RBE Microscope using N FLUOTAR 40⫻ lenses and Leica DMLD camera. Negative control experiments were performed by omitting the primary antibody and using the secondary antibody alone with ␣-bungarotoxin. C2C12 myotubes on slide flasks were rinsed in PBS, fixed for 15 min in PBS containing 2% w/v paraformaldehyde and 1.5% w/v sucrose, washed three times in PBS, permeabilized in 0.5% v/v Triton X-100 in PBS for 10 min and finally washed three times in PBS. Coverslips were processed for immunofluorescence microscopy as described above using the primary antibody ␣1CT-FP (1/1000) or ␣2-PEP (1/100). Immunoblotting Dissected normal C57 and mdx mouse skeletal muscle, heart, and brain were homogenized in 4 ml treat-

ment buffer (75 mM Tris–HCl, pH 6.8, 3.8% w/v SDS, 4 M urea, 20% v/v glycerol). Twenty micrograms of total protein from each tissue was separated on 8% SDS– polyacrylamide gels and transferred onto nitrocelluose membranes (Schleicher and Schuell). Membranes were incubated for 1 h in blocking buffer (Tris-buffered saline containing 0.1% v/v Tween-20 and 5% w/v nonfat dry milk) and incubated for 1 h with the antibody ␣-Pan (1:10). Membranes were washed twice for 5 min in Tris-buffered saline and twice in blocking buffer and incubated for 1 h with HRP-conjugated donkey antirabbit IgG (diluted 1:3000; Jackson ImmunoResearch Laboratories, Inc.). Western blots were developed with the BM chemiluminescence substrate system according to the manufacturer’s instructions (Roche) and exposed to film. In Situ Hybridization Longitudinal serial cryostat sections (12 ␮m) of hind-limb skeletal muscles from C57 mice were mounted onto alternate Superfrost Plus slides (Fisher Scientific) and immediately fixed in 4% w/v paraformaldehyde for 10 min. Alternate slides were then either processed for acetylcholinesterase (AChE) histochemistry to visualize NMJs (Karnovsky and Roots, 1964) or subjected to in situ hybridization as described previously (Gramolini et al., 1997), using synthetic oligonucleotides designed to specifically detect transcripts for each of the ␣-dystrobrevin isoforms -1, -2, and -3. The sequences of the antisense oligonucleotides generated were: ␣ -dystrobrevin-1 (DB1), 5⬘-TAGTTTAATCACTTCAAAATATAACAGTCCCAGGAGGTTCCAAC-3⬘, ␣-dystrobrevin-2 (DB2), 5⬘CGGTGTACAGCTTCTTCTGCTTGCTTCTGAACCTGACTACAAGGTCA-3⬘ and ␣-dystrobrevin-3 (DB3), 5⬘-CAGCACCCTAAAAACAGAAAGTAAAGCCCAGTCTTGACAGTGA-3⬘. The corresponding sense oligonucleotides were generated in each case as control probes (Sigma Genosys). Three independent in situ experiments were performed with each probe. Analysis of in situ hybridization labeling was performed using an image analysis system equipped with Northern Eclipse software (Empix Imaging Inc). The labeling density in synaptic verses extrasynaptic regions was determined by measuring the pixel density within a circular field encompassing the junctional region. The junctional areas were determined by the staining of the serial section for AChE histochemistry. To determine the extrajunctional levels of expression, similar circular fields were quantitated within the same muscle fibre at a site distant from the junctional area. These regions were carefully selected to ensure the

138 absence of large blood vessels and NMJs. A total of 50 measurements were performed for each probe. For these experiments, background values were determined to be the pixel density of regions external to the muscle fibres, i.e., the values obtained from the blank slide, and these values were subtracted from all measurements.

RT-PCR 10 micrograms of total RNA isolated from mouse skeletal muscle and brain was converted into first strand cDNA using AMV Reverse Transcriptase XL (Kramel Biotech) according to the manufacturer’s instructions. Two microliters of first strands was used in 50 ␮l PCR reactions containing 5 ␮l 10⫻ Pfu Turbo reaction buffer (Stratagene), 200 ␮M each dNTPs, 0.5 ␮M forward primer, 0.5 ␮M reverse primer, and 2 U Pfu Turbo DNA polymerase (Stratagene). Twenty cycles of PCR were performed using the following cycling conditions: 1 ⫻ 94°C for 1 min, 20 ⫻ (94°C for 1 min, primer annealing temp for 1 min, 72°C for 2.5 min), 1 ⫻ 72°C for 10 min. Forward primers were located in the 5⬘UTR exons downstream of promoters A, B and C (UTRAf 5⬘-CGGAAGAGTTAGAGGCATGTTG-3⬘; UTRBf 5⬘GGTGACACAGGCGCCGGTCC-3⬘; UTRCf 5⬘-TGGCTAAATCTGTTCTCCCATG-3⬘) and reverse primers were designed to the unique C-terminus of the three ␣-dystrobrevin isoforms (DB1r 5⬘-AGGCAGATGCTGAACGGATG-3⬘; DB2r 5⬘-AGCAATGAGAAGGTCAGCAGGAC-3⬘; DB3r 5⬘-TCATGTTATCCATCTAGACGC-3⬘). Using a combination of the forward and reverse primers, PCR reactions were performed to detect each of the ␣-dystrobrevin isoforms originating from the three promoters in skeletal muscle and brain. Five microliters of each PCR product was dot blotted onto Hybond N⫹ (Amersham Pharmacia Biotech) membrane and probed with a 1.25-kb BglII–HindIII fragment of the m32 cDNA that detects all ␣-dystrobrevin isoforms (Holzfeind et al., 1999).

ACKNOWLEDGMENTS We thank Markus A. Ruegg for his kind gift of C95 A4B8 agrin. This work was generously supported by grants from the Wellcome Trust and the Muscular Dystrophy Association of America. S.E.N. is a Wellcome Trust Prize Student and D.J.B. is a Wellcome Trust Career Development Fellow. A.O.G. is supported by a Ministry of Ontario Graduate Scholarship. B.J.J. is a scientist of the Medical Research Council of Canada. The authors thank Allyson Potter for her expert technical assistance and Colin Akerman for his help with the statistical analysis.

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