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Dystrophin and the α-dystrobrevins bind directly to the adaptor protein syntrophin to form membrane-associated scaffolds. At the blood-brain barrier, α-syntrophin co-localizes with dystrophin and the α-dystrobrevins in perivascular glial endfeet and is required for localization of the water channel aquaporin-4. We have previously shown that localization of the scaffolding proteins γ2-syntrophin, α-dystrobrevin-2, and dystrophin to glial endfeet is also dependent upon the presence of α-syntrophin. In the present study, we show that the expression levels of α-syntrophin, γ2-syntrophin, and dystrophin at the blood-brain barrier are reduced in α-dystrobrevin-null mice. This is the first demonstration that assembly of an astroglial protein scaffold containing syntrophin and dystrophin in perivascular astrocytes is dependent upon the presence of α-dystrobrevin.
Since the discovery of dystrophin as the product of the Duchenne muscular dystrophy gene, numerous proteins have been identified which associate with dystrophin to form membrane-based scaffolds [reviewed in refs. 1 and 2]. These dystrophin-associated scaffolds provide structural integrity to the cell by connecting the extracellular matrix with the cytoskeleton and create intracellular docking points for regulatory proteins. The dystrobrevins are a family of proteins that interact directly with dystrophin and syntrophin adaptor proteins providing a link to a network of signaling proteins that regulate key cellular processes such as water transport and nitric-oxide-mediated events [3–5] Two separate dystrobrevin genes named α- and β-dystrobrevin have been identified, both of which yield multiple transcripts [6–10]. α- and β-dystrobrevin are both expressed in brain but have distinct locations .
The α-dystrobrevin isoforms (currently six have been identified) have been characterized most thoroughly in muscle where several are required for proper formation of the neuromuscular synapse . However, the dystrobrevins are also present in the brain where they are present in both neurons and glia[10–16]. Immunofluorescence studies show αDb at glial endfeet and endothelial cells at the blood-brain barrier [13,15], whereas αDb-1 and -2 localize specifically to glial endfeet [12,16]. We have previously shown that the localization of αDb-2 to glial endfeet at the blood brain barrier is specifically dependent upon the presence of α-syntrophin . Using the α-dystrobrevin-null mouse, we now show that the expression levels of α-syntrophin, γ2-syntrophin, and dystrophin in the vasculature are dependent on α-dystrobrevin. These data indicate that α-syntrophin and α-dystrobrevin are mutually dependent on each other and that α-dystrobrevin plays a critical role in assembly of the DAPC at the blood-brain barrier.
The syntrophin antibodies used in this study were mAb1351 (Affinity Bioreagents; MA1-745), which recognizes α-, β1-, and β2-syntrophin , and syntrophin-isoform specific affinity-purified rabbit polyclonal antibodies to α-syntrophin (Syn17; ) and γ2-syntrophin (Ab 212; [16,18,19]). The dystrophin (MANDRA-mouse monoclonal) was purchased from Sigma (St. Louis, MO) and the α-dystrobrevin antibodies (Ab 638, Ab αDB2-rabbit polyclonals) have been characterized previously [20,21].
For immunofluorescence, animals were euthanized in accordance with the NIH guide for the care of laboratory animals. After light sedation with CO2 gas, 6-week and 3-month-old male C57Bl6 and α-dystrobrevin-null mice  and corresponding wild type littermates were quickly euthanized and brains were dissected. Cerebella were removed, bisected and placed in PBS containing 4% paraformaldehyde/10% sucrose for 12 min on ice. Cerebellar tissue was then transferred to PBS containing 25% sucrose and placed at 4°C overnight. Tissue was embedded in OCT and frozen in isopentane cooled with liquid nitrogen. Twenty µm thick cryosections were adhered to superfrost plus slides (VWR) and stored at −80°C. Immunoflurescence and confocal images were collected under identical conditions, as described previously .
The assembly of the dystrophin-associated protein complex at the blood-brain barrier is dependent upon the presence of α-syntrophin . Specifically, α-syntrophin is required for the localization of γ2-syntrophin, α-dystrobrevin-2, and dystrophin to the perivascular astrocyte endfoot membrane. Since localization of αDB-2, but not αDB-1, requires α-syntrophin, we questioned how the DAPC would be affected by the loss of α-dystrobrevin isoforms. We performed immunolocalization for α-syntrophin on brains from α-dystrobrevin null animals [Figure 1D–F; 22] and wild-type littermates (Figure 1A–C). The merged image in Figure 1C indicates that, in wild type mice, α-syntrophin co-localizes with α-dystrobrevin 1 in perivascular astrocyte endfeet, as reported previously. Furthermore, we observed a striking decrease in the level of α-syntrophin immunofluorescence in the α-dystrobrevin-null mice (compare 1E to 1B).
We have previously shown that the localization of γ2-syntrophin to perivascular endfeet is dependent on α-syntrophin . Since we find that expression of α-syntrophin at glial endfeet is dependent on the presence of α-dystrobrevin, we determined whether the localization of γ2-syntrophin was disrupted by the loss of α-dystrobrevin. We compared γ2-syntrophin immunolocalization (green, Fig 2B, E) in wild-type (Figure 2A–C) and α-dystrobrevin-null (Figure 2D–F) cerebellum. γ2-Syntrophin immunofluorescence was reduced in the surrounding cerebellar vasculature in the absence of α-dystrobrevin (compare 2E to 2B). As in the α-syntrophin null animals , γ2-syntrophin expression is unaffected in neurons (see arrows, Figure 2). Taken together, these data indicate that expression of γ2-syntrophin in non-neuronal cells is dependent on both α-syntrophin and α-dystrobrevin.
Dystrophin colocalizes with α-syntrophin in both the granular layer and the molecular layer of the cerebellum. The α-Db-1 and -2 isoforms exhibit discrete distributions in the vasculature of these layers but both colocalize with α-sybtrophin. Thus, dystrophin and a form of α-dystrobrevin are colocalized in the vasculature throughout the cerebellum. Dystrophin localization at the blood-brain barrier is contingent on the presence of α-syntrophin . Since α-syntrophin localization is disrupted in the α-dystrobrevin-null mice, the localization of dystrophin might also be affected. We assessed immunofluorescence for dystrophin (green, Fig 3B, E) and αDb-2 (red, Fig 3A, D) in cerebellum from wild type (Fig 3A–C) and α-dystrobrevin-null mice (Fig 3D–F). As shown previously, αDb-2 is present in the granular layer of cerebellar cortex (Fig 3A, see arrows). As expected, the α-dystrobrevin-null cerebellum is negative for α-Db2 (Fig 3D). Dystrophin is associated with the blood-brain barrier throughout the cerebellar cortex (Fig 3B), and its expression levels are substantially reduced in the α-dystrobrevin-null mouse (compare 3E to 3B).
Taken together with our previous published results , the main finding of this work is that α-syntrophin and α-dystrobrevin are mutually dependent upon one another for their expression and/or localization in the cerebrovasculature. Our earlier conclusion  that α-syntrophin is the central regulator of the DAPC at endfeet must now be modified to include the α-dystrobrevin/α-syntrophin subcomplex. Whether other syntrophin isoforms are required remains to be determined. Interestingly, co-dependence of α-syntrophin and α-dystrobrevin occurs also in skeletal muscle fibers. In mice lacking α-syntrophin, sarcolemmal staining intensity for αDB-1 and αDB-2 is also decreased . Likewise, in α-dystrobrevin-null mice, levels of α-, β1- and β2-syntrophin were also decreased [11,22].
How might these protein members of the dystrophin complex be mutually dependent on each other for proper localization? One possibility is that they are assembled into a vesicular complex [24,25] prior to transport to glial endfeet. In this capacity, loss of either protein may have effects downstream in the trafficking pathway. Alternatively, α-syntrophin and α-dystrobrevin may act in concert to retain components of the DAPC at the cellular plasma membrane. Future experiments using endothelial-astrocyte co-cultures from normal and α-syntrophin null mice may be informative in revealing the dynamics of DAPC complex trafficking, assembly, and stability at the blood-brain interface.
Loss of either α-syntrophin or α-dystrobrevin results in a reduction of dystrophin from the vasculature. In skeletal muscle, dystrophin is considered the central component of the dystrophin-associated protein complex whose presence is critical for sarcolemmal localization of α-dystrobrevins and certain syntrophin isoforms [11,22,23]. The abence of α-dystrobrevins and syntrophins do not affect sarcolemmal dystrophin levels. Thus, despite some similarities discussed above, assembly and/or maintenance of this dystrophin-associated subcomplex may be regulated differentially in brain and skeletal muscle.
We show for the first time that the loss of α-dystrobrevin results in the decreased expression of α-syntrophin, γ2-syntrophin, and dystrophin in astrocytes. Taken together with our previous study , these data indicate that α-syntrophin and α-dystrobrevin are both essential for the proper assembly and maintenance of the dystrophin-based scaffold in the cerebrovasculature. Therefore, altered expression of either of these proteins could compromise both the structural and signaling functions of this scaffold.
We thank Drs. Stephan Kroger and Mahmood Amiry-Moghaddam for advice regarding brain immunofluorescence protocols, Drs Marv Adams and Ruth Westenbroek for assistance with mouse perfusions, Greg Martin for assistance with confocal imaging, Yan Tesch and Kendra Anderson for their help with mouse husbandry and genotyping, and Charles Mahan for preparation of cryosections. We also thank Drs. Mark Grady and Joshua Sanes of Washington University, St Louis, MO, and Harvard University, Cambridge, MA, respectively, for generously providing α-dystrobrevin-null animals.
Sources of Support: NIH grant RO1 NS33145 to SCF, NIH training grant T32 NS007332 to ADB and SSD and NIH training grant T32 EB0001650 to SSD
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