Marvin E. Adams

An α-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain

The water channel AQP4 is concentrated in perivascular and subpial membrane domains of brain astrocytes. These membranes form the interface between the neuropil and extracerebral liquid spaces. AQP4 is anchored at these membranes by its carboxyl terminus to α-syntrophin, an adapter protein associated with dystrophin. To test functions of the perivascular AQP4 pool, we studied mice homozygous for targeted disruption of the gene encoding α-syntrophin (α-Syn−/−). These animals show a marked loss of AQP4 from perivascular and subpial membranes but no decrease in other membrane domains, as judged by quantitative immunogold electron microscopy. In the basal state, perivascular and subpial astroglial end-feet were swollen in brains of α-Syn−/− mice compared to WT mice, suggesting reduced clearance of water generated by brain metabolism. When stressed by transient cerebral ischemia, brain edema was attenuated in α-Syn−/− mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but α-syntrophin was retained in perivascular astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus α-syntrophin-dependent anchoring of AQP4 is sensitive to ischemia, and loss of AQP4 from this site may retard the dissipation of postischemic brain edema. These studies identify a specific, syntrophin-dependent AQP4 pool that is expressed at distinct membrane domains and which mediates bidirectional transport of water across the brain–blood interface. The anchoring of AQP4 to α-syntrophin may be a target for treatment of brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule.

Syntrophin-dependent expression and localization of Aquaporin-4 water channel protein

The Aquaporin-4 (AQP4) water channel contributes to brain water homeostasis in perivascular astrocyte endfeet where it is concentrated. We postulated that AQP4 is tethered at this site by binding of the AQP4 C terminus to the PSD95-Discs large-ZO1 (PDZ) domain of syntrophin, a component of the dystrophin protein complex. Chemical cross-linking and coimmunoprecipitations from brain demonstrated AQP4 in association with the complex, including dystrophin, β-dystroglycan, and syntrophin. AQP4 expression was studied in brain and skeletal muscle of mice lacking α-syntrophin (α-Syn−/−). The total level of AQP4 expression appears normal in brains of α-Syn−/− mice, but the polarized subcellular localization is reversed. High-resolution immunogold analyses revealed that AQP4 expression is markedly reduced in astrocyte endfeet membranes adjacent to blood vessels in cerebellum and cerebral cortex of α-Syn−/− mice, but is present at higher than normal levels in membranes facing neuropil. In contrast, AQP4 is virtually absent from skeletal muscle in α-Syn−/− mice. Deletion of the PDZ-binding consensus (Ser-Ser-Val) at the AQP4 C terminus similarly reduced expression in transfected cell lines, and pulse–chase labeling demonstrated an increased degradation rate. These results demonstrate that perivascular localization of AQP4 in brain requires α-Syn, and stability of AQP4 in the membrane is increased by the C-terminal PDZ-binding motif.

Delayed K+ clearance associated with aquaporin-4 mislocalization: Phenotypic defects in brains of α-syntrophin-null mice

Recovery from neuronal activation requires rapid clearance of potassium ions (K+) and restoration of osmotic equilibrium. The predominant water channel protein in brain, aquaporin-4 (AQP4), is concentrated in the astrocyte end-feet membranes adjacent to blood vessels in neocortex and cerebellum by association with α-syntrophin protein. Although AQP4 has been implicated in the pathogenesis of brain edema, its functions in normal brain physiology are uncertain. In this study, we used immunogold electron microscopy to compare hippocampus of WT and α-syntrophin-null mice (α-Syn-/-). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the α-Syn-/- mice, whereas labeling of the inwardly rectifying K+ channel, Kir4.1, is largely unchanged. Activity-dependent changes in K+ clearance were studied in hippocampal slices to test whether AQP4 and K+ channels work in concert to achieve isosmotic clearance of K+ after neuronal activation. Microelectrode recordings of extracellular K+ ([K+]o) from the target zones of Schaffer collaterals and perforant path were obtained after 5-, 10-, and 20-Hz orthodromic stimulations. K+ clearance was prolonged up to 2-fold in α-Syn-/- mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the α-Syn-/-mice. These studies lead us to propose that water flux through perivascular AQP4 is needed to sustain efficient removal of K+ after neuronal activation.

rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice.

Mice carrying mutations in both the dystrophin and utrophin genes die prematurely as a consequence of severe muscular dystrophy. Here, we show that intravascular administration of recombinant adeno-associated viral (rAAV) vectors carrying a microdystrophin gene restores expression of dystrophin in the respiratory, cardiac and limb musculature of these mice, considerably reducing skeletal muscle pathology and extending lifespan. These findings suggest rAAV vector–mediated systemic gene transfer may be useful for treatment of serious neuromuscular disorders such as Duchenne muscular dystrophy.

Alpha-syntrophin deletion removes the perivascular but not endothelial pool of aquaporin-4 at the blood–brain barrier and delays the development of brain edema in an experimental model of acute hyponatremia

The formation of brain edema, commonly occurring as a potentially lethal complication of acute hyponatremia, is delayed following knockout of the water channel aquaporin‐4 (AQP4). Here we show by high‐resolution immunogold analysis of the blood–brain‐barrier that AQP4 is expressed in brain endothelial cells as well as in the perivascular membranes of astrocyte endfeet. A selective removal of perivascular AQP4 by α‐syntrophin deletion delays the buildup of brain edema (assessed by Diffusion‐weighted MRI) following water intoxication, despite the presence of a normal complement of endothelial AQP4. This indicates that the perivascular membrane domain, which is peripheral to the endothelial blood–brain barrier, may control the rate of osmotically driven water entry. This study is also the first to demonstrate that the time course of edema development differs among brain regions, probably reflecting differences in aquaporin‐4 distribution. The resolution of the molecular basis and subcellular site of osmotically driven brain water uptake should help design new therapies for acute brain edema.

Two forms of mouse syntrophin, a 58 kd dystrophin-associated protein, differ in primary structure and tissue distribution.

Syntrophin, a 58 kd extrinsic membrane protein, is concentrated at postsynaptic sites at the neuromuscular junction and may be involved in clustering acetylcholine receptors. In muscle and nonmuscle tissues, syntrophin is associated with dystrophin, utrophin, and two homologs of the dystrophin carboxy-terminal region. We have isolated three cDNAs encoding Torpedo and mouse syntrophins. The Torpedo cDNA encodes a full-length protein, and on Northern blots recognizes a 3.5 kb mRNA. The two mouse syntrophin cDNAs are products of separate genes but encode proteins that share 50% identity. Syntrophin-1 mRNA (2.2 kb) is expressed at highest levels in skeletal muscle. Syntrophin-2 mRNAs (2.2, 5.0, and 10 kb) are expressed in all mouse tissues examined. These patterns of expression suggest that syntrophin-1 and syntrophin-2 may associate with different members of the dystrophin family.

Protein Trafficking and Anchoring Complexes Revealed by Proteomic Analysis of Inward Rectifier Potassium Channel (Kir2.x)-associated Proteins

Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including α1-, β1-, and β2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.

The Potassium Channel Kir4.1 Associates with the Dystrophin-Glycoprotein Complex via α-Syntrophin in Glia

One of the major physiological roles of potassium channels in glial cells is to promote “potassium spatial buffering” in the central nervous system, a process necessary to maintain an optimal potassium concentration in the extracellular environment. This process requires the precise distribution of potassium channels accumulated at high density in discrete subdomains of glial cell membranes. To obtain a better understanding of how glial cells selectively target potassium channels to discrete membrane subdomains, we addressed the question of whether the glial inwardly rectifying potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex (DGC). Immunoprecipitation experiments revealed that Kir4.1 is associated with the DGC in mouse brain and cultured cortical astrocytes. In vitro immunoprecipitation and pull-down assays demonstrated that Kir4.1 can bind directly to α-syntrophin, requiring the presence of the last three amino acids of the channel (SNV), a consensus PDZ domain-binding motif. Furthermore, Kir4.1 failed to associate with the DGC in brains from α-syntrophin knockout mice. These results suggest that Kir4.1 is localized in glial cells by its association with the DGC through a PDZ domain-mediated interaction with α-syntrophin and suggest an important role for the DGC in central nervous system physiology.

In vivo requirement of the α-syntrophin PDZ domain for the sarcolemmal localization of nNOS and aquaporin-4

α-Syntrophin is a scaffolding adapter protein expressed primarily on the sarcolemma of skeletal muscle. The COOH-terminal half of α-syntrophin binds to dystrophin and related proteins, leaving the PSD-95, discs-large, ZO-1 (PDZ) domain free to recruit other proteins to the dystrophin complex. We investigated the function of the PDZ domain of α-syntrophin in vivo by generating transgenic mouse lines expressing full-length α-syntrophin or a mutated α-syntrophin lacking the PDZ domain (ΔPDZ). The ΔPDZ α-syntrophin displaced endogenous α- and β1-syntrophin from the sarcolemma and resulted in sarcolemma containing little or no syntrophin PDZ domain. As a consequence, neuronal nitric oxide synthase (nNOS) and aquaporin-4 were absent from the sarcolemma. However, the sarcolemmal expression and distribution of muscle sodium channels, which bind the α-syntrophin PDZ domain in vitro, were not altered. Both transgenic mouse lines were bred with an α-syntrophin–null mouse which lacks sarcolemmal nNOS and aquaporin-4. The full-length α-syntrophin, not the ΔPDZ form, reestablished nNOS and aquaporin-4 at the sarcolemma of these mice. Genetic crosses with the mdx mouse showed that neither transgenic syntrophin could associate with the sarcolemma in the absence of dystrophin. Together, these data show that the sarcolemmal localization of nNOS and aquaporin-4 in vivo depends on the presence of a dystrophin-bound α-syntrophin PDZ domain.

MOUSE ALPHA 1- AND BETA 2-SYNTROPHIN GENE STRUCTURE, CHROMOSOME LOCALIZATION, AND HOMOLOGY WITH A DISCS LARGE DOMAIN

The syntrophin family of dystrophin-associated proteins consists of three isoforms, α1, β1, and β2, each encoded by a distinct gene. We have cloned and characterized the mouse α1- and β2-syntrophin genes. The mouse α1-syntrophin gene (>24 kilobases) is comprised of eight exons. The mouse β2-syntrophin gene (>33 kilobases) contains seven exons, all of which have homologues at the corresponding position in the α1-syntrophin gene. Primer extension analysis reveals two transcription initiation sites in the α1-syntrophin gene and a single site in the β2-syntrophin gene. The sequence immediately 5′ of the transcription start sites of both genes lacks a TATA box but is GC-rich and has multiple putative SP1 binding sites. The α1-syntrophin gene is located on human chromosome 20 and mouse chromosome 2, while the β2-syntrophin gene is on human chromosome 16 and mouse chromosome 8. Analysis of the amino acid sequence of the syntrophins reveals the presence of four conserved domains. The carboxyl-terminal 56 amino acids are highly conserved and constitute a syntrophin unique domain. Two pleckstrin homology domains are located at the amino-terminal end of the protein. The first pleckstrin homology domain is interrupted by a domain homologous to repeated sequences originally found in the Drosophila discs-large protein.