α-Dystrobrevin associates with and is a homologue of dystrophin, the protein linked to Duchenne and Becker muscular dystrophies. We used a transgenic approach to restore α-dystrobrevin to the sarcolemma in mice that lack dystrophin (mdx mice) to study two interrelated functions: (1) the ability of α-dystrobrevin to rescue components of the dystrophin complex in the absence of dystrophin and (2) the ability of sarcolemmal α-dystrobrevin to ameliorate the dystrophic phenotype. We generated transgenic mice expressing α-dystrobrevin-2a linked to a palmitoylation signal sequence and bred them onto the α-dystrobrevin-null and mdx backgrounds. Expression of palmitoylated α-dystrobrevin prevented the muscular dystrophy observed in the α-dystrobrevin-null mice, demonstrating that the altered form of α-dystrobrevin was functional. On the mdx background, the palmitoylated form of α-dystrobrevin was expressed on the sarcolemma but did not significantly ameliorate the muscular dystrophy phenotype. Palmitoylated dystrobrevin restored α-syntrophin and aquaporin-4 (AQP4) to the mdx sarcolemma but was unable to recruit β-dystroglycan or the sarcoglycans. Despite restoration of sarcolemmal α-syntrophin, neuronal nitric oxide synthase (nNOS) was not localized to the sarcolemma, suggesting that nNOS requires both dystrophin and α-syntrophin for correct localization. Thus, although nNOS and AQP4 both require interaction with the PDZ domain of α-syntrophin for sarcolemmal association, their localization is regulated differentially.
In skeletal muscle, the absence of a functional form of the protein dystrophin results in severe muscular dystrophy (Hoffman et al., 1987). Dystrophin localizes to the sarcolemma and stabilizes a complex of proteins (dystrophin-associated proteins, DAPs) that link the extracellular matrix to the actin cytoskeleton (Ohlendieck, 1996). In Duchenne muscular dystrophy (DMD) patients or in dystrophic animal models, the loss of dystrophin leads to the loss or reduction of sarcolemmal DAPs, including β-dystroglycan, the sarcoglycans, α-dystrobrevin, syntrophins, aquaporin-4 (AQP4) and neuronal nitric oxide synthase (nNOS) (Brenman et al., 1995; Ervasti et al., 1990; Frigeri et al., 1998).
Among the DAPs, α-dystrobrevin is unique because it not only binds dystrophin but it also shares significant homology with dystrophin (Wagner et al., 1993; Yoshida et al., 1995). α-Dystrobrevin is alternatively spliced to produce five major isoforms. Three isoforms are expressed in skeletal muscle, α-dystrobrevin-1, -2 and -3. α-Dystrobrevin-1 is most abundant at the postsynaptic neuromuscular junctions although it is also expressed to some degree on the sarcolemma of some fibers (Nawrotzki et al., 1998). It is α-dystrobrevin-2a that is most abundant on the sarcolemma where it colocalizes with dystrophin. α-Dystrobrevin-2b has a unique 11 amino-acid-long C-terminus and is also found in skeletal muscle at low levels (Enigk and Maimone, 1999). Little is known about the localization of α-dystrobrevin-3, a short isoform that lacks the dystrophin and syntrophin interaction sites. Mice lacking these isoforms of α-dystrobrevin have a muscular dystrophy but it is less severe than that caused by the absence of dystrophin (Grady et al., 1999).
Several studies have shown that α-dystrobrevin can bind many of the same proteins that associate with dystrophin. Co-purification studies indicate that dystrobrevin interacts directly with sarcoglycans (Yoshida et al., 2000). The evidence for sarcoglycan association is strengthened by the observation that patients with limb girdle muscular dystrophy, caused by the loss of sarcoglycans, have reduced levels of sarcolemmal α-dystrobrevin (Metzinger et al., 1997). Studies in retina have shown an interaction between α-dystrobrevin and β-dystroglycan (Claudepierre et al., 2000). Finally, both dystrophin and α-dystrobrevin contain two binding sites for syntrophin (Newey et al., 2000), an adapter protein that links signaling proteins to the dystrophin-dystrobrevin scaffold. Proteins associated with syntrophin include kinases (Hogan et al., 2001; Lumeng et al., 1999), ion channels (Connors et al., 2004; Gee et al., 1998; Leonoudakis et al., 2004), water channels (Neely et al., 2001) and nNOSμ (Brenman et al., 1996). Therefore, both dystrophin and dystrobrevin serve as scaffolds for a variety of signaling proteins.
Homology and protein-binding similarities have led to the idea that dystrobrevin partially compensates for the loss of dystrophin in dystrophic muscle. This idea is supported by studies in C. elegans, which show that overexpression of dystrobrevin (dyb-1) delays the onset of myopathy in the dystrophin-MyoD (dys-1;hlh-1) double mutant (Gieseler et al., 2002). However, when α-dystrobrevin is overexpressed in muscle of mice that lack dystrophin (mdx mice), the α-dystrobrevin fails to accumulate on the sarcolemma (Grady et al., 2003). To overcome this problem, we altered α-dystrobrevin by introduction of a palmitoylation site to force sarcolemmal localization of α-dystrobrevin-2 in dystrophin-deficient mouse muscle. This allowed us to test whether sarcolemmal dystrobrevin can restore DAPs to the sarcolemma in the absence of dystrophin. We identified a subset of DAPs that are rescued by expression of sarcolemmal α-dystrobrevin.
In normal muscle, α-dystrobrevin associates directly with the C-terminal region of dystrophin and plays an important role in the signaling function of the dystrophin protein complex. Transgenically expressed α-dystrobrevin in skeletal muscle does not accumulate at the sarcolemma in the absence of dystrophin (Grady et al., 2003). We reasoned that targeting of dystrobrevin to the sarcolemma in the absence of dystrophin may be sufficient to stabilize all or part of the DAP complex and thereby potentially ameliorate the resulting muscular dystrophy.
To facilitate targeting of dystrobrevin to the sarcolemma, we attached the palmitoylation signal sequence from the murine K-Ras oncogene (Kahn et al., 1987) to the C-terminus of α-dystrobrevin-2a, the main sarcolemmal isoform. We also made a construct with only an hemagglutinin (HA) tag at the C-terminus to serve as a control for altering the dystrobrevin C-terminus. Both constructs were placed under the control of the striated-muscle-specific creatine kinase promoter and were used to generate lines of transgenic mice. We obtained three founder lines expressing α-dystrobrevin linked to HA only (TgDB) and five lines (TgDB-RPS) that expressed dystrobrevin with the HA tag and the Ras palmitoylation sequence (RPS). We selected lines expressing high and low levels of each transgene product (TgDB-63, TgDB-68, TgDB-RPS-29, TgDB-RPS-71) for breeding onto both dystrobrevin-null and mdx backgrounds (Fig. 1).
Transgene expression on the α-dystrobrevin-null background
The transgenic lines were bred onto the α-dystrobrevin-null background to test the functionality of the altered α-dystrobrevins. Both constructs have altered C-termini that could potentially affect their function. We assessed the ability of the α-dystrobrevin transgenes to reduce the number of central nuclei (a marker for muscle fiber regeneration) and lower the levels of serum creatine kinase (a measure of sarcolemmal integrity) (Fig. 2). Central nuclei counts were performed on a primarily fast-twitch muscle (tibialis anterior), a primarily slow-twitch muscle (soleus) and on the diaphragm muscle. Results from all three muscles indicate that expression of each transgene was able to restore central nuclei counts to near wild type levels in the α-dystrobrevin-null mice (Fig. 2B). Likewise, serum creatine kinase levels were also reduced to near wild type levels with each transgene (Fig. 2C). These data indicate that the addition of an HA tag and the palmitoylation signal sequence does not interfere with the ability of α-dystrobrevin to prevent dystrophy.
We also assessed the ability of the transgenic α-dystrobrevin to restore normal sarcolemmal association of α-syntrophin and neuronal nitric oxide synthase (nNOS), which is reduced in muscle of the α-dystrobrevin-null mice (Grady et al., 1999). As expected, both transgenic α-dystrobrevins localized correctly to the sarcolemma (Fig. 3). Furthermore, both transgenic α-dystrobrevins restored sarcolemmal levels of α-syntrophin and nNOS. The TgDB-RPS transgene appeared to recruit slightly greater amounts of α-syntrophin and nNOS to the sarcolemma, but this is most probably owing to higher expression of this transgene compared with that of the TgDB line (line 63 compared with line 29 in Fig. 1). These data indicate that the TgDB-RPS protein can functionally compensate for the lack of α-dystrobrevin. We therefore proceeded to test whether this transgene can attenuate the dystrophic phenotype of the mdx mouse.
Transgene expression on mdx background
Transgenic mice that expressed α-dystrobrevin with and without the palmitoylation signal sequence were bred with mdx mice to express α-dystrobrevin in the absence of dystrophin. Again, we assessed the ability of the transgenic α-dystrobrevin to attenuate the dystrophic mdx phenotype by determining the number of central nuclei in the tibialis anterior, soleus and diaphragm muscles (Fig. 4). Mice expressing transgenic α-dystrobrevin did not have significantly fewer centrally nucleated muscle fibers than mdx mice (Fig. 4B). A slight trend towards fewer central nuclei in the diaphragm was observed in the TgDB-RPS mice, but this difference was not significant. Likewise, serum creatine kinase levels were similar between mdx mice and TgDB-RPS–mdx mice (Fig. 4C).
To confirm that the TgDB-RPS was targeting to the sarcolemma in the absence of dystrophin, we used HA immunofluorescence to localize the transgenic α-dystrobrevin (Fig. 5A). As expected, the palmitoylated α-dystrobrevin specifically localized to the sarcolemma. Non-palmitoylated dystrobrevin was not present on the sarcolemma, but was found at the neuromuscular junction where high levels of utrophin are expressed (data not shown). Despite being expressed on the sarcolemma, the palmitoylated α-dystrobrevin was unable to increase membrane levels of β-dystroglycan (Fig. 5). Likewise, palmitoylated α-dystrobrevin did not increase the sarcolemmal levels of α-sarcoglycan in the TgDB-RPS/mdx mice (Fig. 5).
The TgDB-RPS was able to increase the levels of sarcolemmal α-syntrophin (Fig. 6). Immunoblotting showed that membrane-associated α-syntrophin levels increased to levels higher than those found in the control mice (strain C57bI6) (Fig. 6B). Despite the high levels of sarcolemmal α-syntrophin, sarcolemmal nNOS levels were not restored in these mice. nNOS is expressed in the cytosol of mdx muscle (Fig. 6) (Thomas et al., 1998) but is not recruited to the sarcolemma by the α-dystrobrevin-bound syntrophin. This provides in vivo evidence that the sarcolemmal localization of nNOS requires both syntrophin and dystrophin. The α-syntrophin recruited to the sarcolemmal by TgDB-RPS was functional, as demonstrated by its ability to restore normal sarcolemmal expression of AQP4. These data demonstrate a fundamental difference in the mechanism by which α-syntrophin regulates nNOS and AQP4 association with the membrane.
In addition to binding dystrophin, α-dystrobrevin can also associate directly with utrophin (Peters et al., 1998). In the α-syntrophin-null mouse, utrophin fails to localize to the neuromuscular junction, suggesting a unique interaction between these two proteins (Adams et al., 2000). We investigated whether the increase in sarcolemmal α-dystrobrevin and α-syntrophin in TgDB-RPS–mdx mice would lead to an increase in the levels of sarcolemmal utrophin. Immunofluorescence studies and immunoblots did not show an increase in membrane-associated utrophin in the α-dystrobrevin transgenics compared to the mdx mouse (Fig. 6).
Dystrophin is thought to function both as a structural link between the extracellular matrix and the cytoskeleton, and as a signaling scaffold. We hypothesized that α-dystrobrevin can perform similar functions if correctly localized to the sarcolemma. Structurally, α-dystrobrevin can potentially link the transmembrane proteins, β-dystroglycan and the sarcoglycans (Claudepierre et al., 2000; Yoshida et al., 2000), with the intermediate filament cytoskeleton by interacting with synemin or syncoilin (Mizuno et al., 2001; Newey et al., 2001). As a signaling scaffold, α-dystrobrevin could bind syntrophins and restore the α-syntrophin-associated proteins nNOS and AQP4. To test these ideas, we generated a transgenic mouse expressing a palmitoylated form of α-dystrobrevin in skeletal muscle.
The palmitoylated α-dystrobrevin transgenic line was initially bred onto the dystrobrevin-null background to determine whether the altered form of α-dystrobrevin was able to rescue the dystrobrevin-null phenotype. Immunofluorescence studies using antibodies against the HA tag showed that palmitoylated α-dystrobrevin was correctly localized and restored normal levels of α-syntrophin and nNOS at the sarcolemma. Determination of the number of centrally nucleated muscle fibers showed that expression of the palmitoylated α-dystrobrevin alleviated the muscular dystrophy present in the α-dystrobrevin-null mice. This finding was further corroborated by data showing that serum creatine kinase levels were also restored to normal levels by the presence of the palmitoylated dystrobrevin. Collectively, these data indicate that the addition of a palmitoylation site at the C-terminus of α-dystrobrevin does not impair its function.
Subsequently, we expressed the palmitoylated α-dystrobrevin on the mdx background and determined whether sarcolemma-targeted α-dystrobrevin restores localization of the dystrophin-associated proteins to dystrophin-deficient muscle. Expression of the TgDB-RPS transgene did not ameliorate the mdx muscular dystrophy, as assessed by serum creatine kinase levels and centrally nucleated fiber counts in three different muscles (Fig. 4).
The palmitoylated form of α-dystrobrevin localized to the sarcolemma in the absence of dystrophin. However, TgDB-RPS was unable to restore detectable levels of either β-dystroglycan or the sarcoglycans. Although there is good evidence that α-dystrobrevin can interact directly with β-dystroglycan and the sarcoglycans (Claudepierre et al., 2000; Yoshida et al., 2000), our data indicate that such interactions are not sufficient to restore sarcolemmal expression of these proteins in the absence of dystrophin. Since the TgDB-RPS is expressed on the sarcolemma at high levels, we predicted that it would bind utrophin and potentially increase the levels of this dystrophin homologue on the sarcolemma. Although the levels of utrophin remained higher than those of wild type in the TgDB-RPS mouse muscle (similar to mdx mice), we did not observe an increase in sarcolemmal utrophin levels compared with those of the mdx mouse. Previous studies have shown that α-dystrobrevin-1 is the isoform that preferentially binds utrophin (Peters et al., 1998). Thus, the failure to enhance sarcolemmal utrophin levels may have resulted, in part, from our use of α-dystrobrevin-2a.
Palmitoylated α-dystrobrevin restored α-syntrophin to the sarcolemma in the absence of dystrophin. The levels of membrane-associated α-syntrophin in TgDB-RPS–mdx mice were even greater than the levels in C57 control mice. We therefore assessed whether the syntrophin-associated proteins AQP4 and nNOS are restored to the muscle membrane of mdx mice by the membrane-targeted α-dystrobrevin.
Sarcolemmal expression of the water channel AQP4 is lost in mdx mice, DMD patients and some Becker muscular dystrophies (BMD) patients (Crosbie et al., 2002; Frigeri et al., 2002; Frigeri et al., 1998). Syntrophin is necessary for sarcolemmal AQP4 expression because mice that lack α-syntrophin (or only the α-syntrophin PDZ domain) fail to express AQP4 on the sarcolemma (Adams et al., 2001; Neely et al., 2001; Yokota et al., 2000). Expression of TgDB-RPS restores AQP4 to the sarcolemma of mdx mice, presumably via interactions with α-syntrophin (Fig. 6). Muscle from some BMD patients and mouse models of BMD lack sarcolemmal AQP4, even though α-syntrophin remains present on the sarcolemma (Crosbie et al., 2002). Thus, α-syntrophin alone is not sufficient for AQP4 localization; other protein interactions must also be important. Dystrophin, dystroglycan, and sarcoglycans are not absolutely required for sarcolemmal localization of AQP4 because membrane-targeted α-dystrobrevin and α-syntrophin are sufficient.
Like AQP4, nNOS expression at the sarcolemma requires the presence of the α-syntrophin PDZ domain (Adams et al., 2000; Adams et al., 2001; Kameya et al., 1999). Several studies have shown a direct interaction between the α-syntrophin PDZ domain and a β-loop structure of nNOS located next to its own PDZ domain (Brenman et al., 1996; Wang et al., 2000). However, despite the ability of nNOS and α-syntrophin to interact with each other in vitro, there are several examples in vivo in which α-syntrophin is present on the sarcolemma but nNOS fails to colocalize. Muscle biopsies from some BMD patients with in-frame deletions in the rod region of dystrophin have normal sarcolemmal α-syntrophin expression but no sarcolemmal nNOS (Chao et al., 1996; Grozdanovic et al., 1997). Also, transgenic mice that express short forms of dystrophin that rescue the mdx dystrophic phenotype often do not restore sarcolemmal nNOS (Judge et al., 2006; Phelps et al., 1995; Yue et al., 2006). The transgenic TgDB-RPS–mdx mice express sarcolemmal syntrophin but are unable to restore sarcolemmal nNOS. Together, these data suggest that correct localization of nNOS requires both α-syntrophin and a portion of dystrophin.
Although absence of the muscle-specific form nNOSμ does not lead to a muscular myopathy, overexpression of the brain isoform nNOSα in mdx muscle ameliorates the dystrophy caused by the loss of dystrophin (Wehling et al., 2001). Sarcolemmal localization of nNOS is also required for normal regulation of muscle blood flow (Thomas et al., 2003). It is likely that disruption of the NO signaling pathway contributes to the severity of the dystrophy that occurs in the absence of dystrophin. Thus, it will be important to determine the precise protein interactions and the regulatory mechanisms that stabilize the expression of nNOS at the skeletal muscle sarcolemma.
Materials and Methods
Rabbit polyclonal antibodies against α-syntrophin, α-dystrobrevin and utrophin have been previously characterized (Blake et al., 1996; Kramarcy et al., 1994; Peters et al., 1997). Rabbit anti-glyceraldehyde-3-phosphate dehydrogenase was purchased from Cell Signaling Technology (Danvers, MA). The polyclonal anti-HA and nNOS antibodies were purchased from Zymed Laboratories, Inc. (San Francisco, CA). The rabbit polyclonal Ab against AQP4 purchased from Chemicon (Temecula, CA) was used for immunofluorescence and a goat polyclonal (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was used for immunoblots. Monoclonal antibodies against β-dystroglycan and α-sarcoglycan were purchased from Vision Biosystems (Norwell, MA).
The cDNA encoding full-length mouse α-dystrobrevin-2a (pSK-m87 16.1A) was a generous gift from Margaret Maimone (SUNY Upstate Medical University, Syracuse, NY). We used polymerase chain reaction (PCR) to add either a single hemagglutinin (HA) epitope tag to the C-terminal of α-dystrobrevin-2a or the HA tag followed by the palmitylation signal sequence from the K-Ras oncogene (amino acid sequence: 5′-KDGKKKKKKSKTKCVIM-3′) (Kahn et al., 1987) (see Fig. 1A). The resulting constructs were cloned into the SacII site of vector pCKVA (Cox et al., 1993), a gift from Jeff Chamberlain and Stephen Hauschka (University of Washington, Seattle, WA). The pCKVA vector contains 3300 bp of the mouse muscle creatine kinase promoter, a 700 bp viral intron and a 200 bp poly-A signal sequence. The linearized constructs were injected into C57Bl6 oocytes (University of Washington Transgenic Facility). Three independent lines of mice harboring the HA only construct (TgDB) and five lines of mice harboring the HA-Ras palmitoylation sequence (TgDB-RPS) were identified by PCR. The TgDB lines and three TgDB-RPS lines were bred onto the dystrobrevin-null and mdx backgrounds. All animal experiments were performed with approval of the Institutional Animal Care and Use Committee at the University of Washington.
Freshly isolated mouse muscle (tibialis anterior, soleus and diaphragm) isolated from adult (16-20 weeks old) mice was flash frozen in liquid-nitrogen-cooled isopentane. Cryostat sections (10 μm) from the mid body of the muscle were stained with haematoxilin and eosin (H&E) and each fiber in the muscle cross section was assessed for central or non-central nuclei using images obtained with a Zeiss Axioskop 2 microscope. The percentage of the fibers containing central nuclei was determined from a minimum of three mice of each genotype. For statistical purposes, each mouse was considered as a single event.
Creatine kinase assay
Blood (50-100 μl) was collected from adult (18-24 weeks old) mice through the saphenous vein (Hem et al., 1998), allowed to clot for 20 minutes, and centrifuged to separate the serum. Serum creatine kinase levels were determined from six to eight mice per genotype using a kit purchased from Stanbio Laboratory (Boerne, TX), according to the manufacturer's protocol.
Immunofluorescence labeling was performed on unfixed adult (16-20 week old) muscle flash frozen in liquid nitrogen cooled isopentane. Cryosections (8 μm) were incubated with antibody as described (Peters et al., 1997). Primary antibodies were visualized using Alexa-Fluor-488-conjugated secondary antibodies (Molecular Probes, Eugene, OR). Sections were co-labeled with Alexa-Fluor-568-conjugated α-bungarotoxin (Molecular Probes) to identify neuromuscular junctions. Images were obtained using a Leica TCS-NT confocal microscope at the W.M. Keck Center for Advanced Studies in Neural Signaling at the University of Washington.
Sarcolemma enrichment and immunoblots
Crude surface membrane was purified from adult (18-week to 24-week old) mouse muscle (whole mouse muscle and bone) as described (Ohlendieck et al., 1991). Protein content of the preparations was determined using the bicinchonic acid assay (Pierce). Gels (4-15% gradient, BioRad) were loaded with 20 μg of protein and the separated proteins transferred to PDVF membrane (Millipore). Primary antibody was detected using HRP conjugated secondary antibody and the Supersignal West Pico substrate (Pierce) with light emission detected by a CCD camera (Alpha Innotech).
We thank Margaret Maimone for providing the α-dystrobrevin-2a cDNA, Joshua Sanes and Mark Grady for use of the α-dystrobrevin knockout mouse, Scott Heximer for providing a clone of the k-ras palmitoylation-signal sequence, and Jeff Chamberlain and Stephen Hauschka for providing the MCK promoter. This work was supported by NIH grant PO1 NS046788, a generous gift from the Apex Foundation, NRSA fellowship F32-NS10900 (D.E.A.), a Parent Project MD fellowship (J.M.P.), and a Mary Gates Fellowship (J.I.C.).
- Accepted October 11, 2007.
- © The Company of Biologists Limited 2008