Biomedical subjects

Stanley C Froehner

Publications and source records attributed to Stanley C Froehner.

At least 19 recordsLinked to original sources

PubMed2006

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.

Animals↗

PubMed2006

Biglycan regulates the expression and sarcolemmal localization of dystrobrevin, syntrophin, and nNOS.

The dystrophin-associated protein complex (DAPC) provides a linkage between the cytoskeleton and the extracellular matrix (ECM) and is also a scaffold for a host of signaling molecules. The constituents of the DAPC must be targeted to the sarcolemma in order to properly function. Biglycan is an ECM molecule that associates with the DAPC. Here, we show that biglycan null mice exhibit a mild dystrophic phenotype and display a selective reduction in the localization of alpha-dystrobrevin-1 and -2, alpha- and beta1-syntrophin, and nNOS at the sarcolemma. Purified biglycan induces nNOS redistribution to the plasma membrane in cultured muscle cells. Biglycan protein injected into muscle becomes stably associated with the sarcolemma and ECM for at least 2 wk. This injected biglycan restores the sarcolemmal expression of alpha-dystrobrevin-1 and -2, and beta1- and beta2-syntrophin in biglycan null mice. We conclude that biglycan is important for the maintenance of muscle cell integrity and plays a direct role in regulating the expression and sarcolemmal localization of the intracellular signaling proteins dystrobrevin-1 and -2, alpha- and beta1-syntrophin and nNOS.

Animals↗

PubMed2006

gamma-Syntrophin scaffolding is spatially and functionally distinct from that of the alpha/beta syntrophins.

The syntrophins are a family of scaffolding proteins with multiple protein interaction domains that link signaling proteins to dystrophin family members. Each of the three most characterized syntrophins (alpha, beta1, beta2) contains a PDZ domain that binds a unique set of signaling proteins including kinases, ion and water channels, and neuronal nitric oxide synthase (nNOS). The PDZ domains of the gamma-syntrophins do not bind nNOS. In vitro pull-down assays show that the gamma-syntrophins can bind dystrophin but have unique preferences for the syntrophin binding sites of dystrophin family members. Despite their ability to bind dystrophin in vitro, neither gamma-syntrophin isoform co-localizes with dystrophin in skeletal muscle. Furthermore, gamma-syntrophins do not co-purify with dystrophin isolated from mouse tissue. These data suggest that the interaction of gamma-syntrophin with dystrophin is transient and potentially subject to regulatory mechanisms. gamma1-Syntrophin is highly expressed in brain and is specifically localized in hippocampal pyramidal neurons, Purkinje neurons in cerebellum, and cortical neurons. gamma2-Syntrophin is expressed in many tissues including skeletal muscle where it is found only in the subsynaptic space beneath the neuromuscular junction. In both neurons and muscle, gamma-syntrophin isoforms localize to the endoplasmic reticulum where they may form a scaffold for signaling and trafficking.

Amino Acid Sequence↗

PubMed2006

Assembly of a perivascular astrocyte protein scaffold at the mammalian blood-brain barrier is dependent on alpha-syntrophin.

alpha-Syntrophin, a member of the dystrophin-associated protein complex, is required for proper localization of the water channel aquaporin-4 at the blood-brain barrier. Mice lacking alpha-syntrophin have reduced levels of aquaporin-4 in perivascular astroglial endfeet. Consequently, they exhibit reduced edema and infarct volume in brain trauma models and reduced K+ clearance from the neuropil, leading to increased seizure susceptibility. We have used the alpha-syntrophin null mice to investigate whether alpha-syntrophin is required for proper localization of other components of the dystrophin complex at the blood-brain barrier. We find that alpha-syntrophin is required for the full recruitment of gamma2-syntrophin and alpha-dystrobrevin-2 to glial endfeet in adult cerebellum. In contrast, the localization of beta1- and beta2-syntrophin and alpha-dystrobrevin-1 at the blood-brain barrier is not dependent on the presence of alpha-syntrophin. The localization patterns of alpha-dystrobrevin-1 and -2 in wild type cerebellum are strikingly different; while alpha-dystrobrevin-1 is present in glial endfeet throughout the cerebellum, alpha-dystrobrevin-2 is restricted to glial endfeet in the granular layer alone. Finally, we show that the enrichment of dystrophin in glial endfeet depends on the presence of alpha-syntrophin. This finding is the first demonstration that dystrophin localization is dependent on syntrophin. Since the localization of gamma2-syntrophin, alpha-dystrobrevin-2, and dystrophin is contingent on alpha-syntrophin, we conclude that alpha-syntrophin is a central organizer of the astrocyte dystrophin complex, an important molecular scaffold for localization of aquaporin-4 at the blood-brain barrier.

Animals↗

PubMed2005

Structure of the split PH domain and distinct lipid-binding properties of the PH-PDZ supramodule of alpha-syntrophin.

Pleckstrin homology (PH) domains play diverse roles in cytoskeletal dynamics and signal transduction. Split PH domains represent a unique subclass of PH domains that have been implicated in interactions with complementary partial PH domains 'hidden' in many proteins. Whether partial PH domains exist as independent structural units alone and whether two halves of a split PH domain can fold together to form an intact PH domain are not known. Here, we solved the structure of the PH(N)-PDZ-PH(C) tandem of alpha-syntrophin. The split PH domain of alpha-syntrophin adopts a canonical PH domain fold. The isolated partial PH domains of alpha-syntrophin, although completely unfolded, remain soluble in solution. Mixing of the two isolated domains induces de novo folding and yields a stable PH domain. Our results demonstrate that two complementary partial PH domains are capable of binding to each other to form an intact PH domain. We further showed that the PH(N)-PDZ-PH(C) tandem forms a functionally distinct supramodule, in which the split PH domain and the PDZ domain function synergistically in binding to inositol phospholipids.

Animals↗

PubMed2005

{alpha}-Syntrophin regulates ARMS localization at the neuromuscular junction and enhances EphA4 signaling in an ARMS-dependent manner.

EphA4 signaling has recently been implicated in the regulation of synapse formation and plasticity. In this study, we show that ankyrin repeat-rich membrane spanning (ARMS; also known as a kinase D-interacting substrate of 220 kD), a substrate for ephrin and neurotrophin receptors, was expressed in developing muscle and was concentrated at the neuromuscular junction (NMJ). Using yeast two-hybrid screening, we identified a PDZ (PSD-95, Dlg, ZO-1) domain protein, alpha-syntrophin, as an ARMS-interacting protein in muscle. Overexpression of alpha-syntrophin induced ARMS clustering in a PDZ domain-dependent manner. Coexpression of ARMS enhanced EphA4 signaling, which was further augmented by the presence of alpha-syntrophin. Moreover, the ephrin-A1-induced tyrosine phosphorylation of EphA4 was reduced in C2C12 myotubes after the blockade of ARMS and alpha-syntrophin expression by RNA interference. Finally, alpha-syntrophin-null mice exhibited a disrupted localization of ARMS and EphA4 at the NMJ and a reduced expression of ARMS in muscle. Altogether, our findings suggest that ARMS may play an important role in regulating postsynaptic signal transduction through the syntrophin-mediated localization of receptor tyrosine kinases such as EphA4.

Animals↗

PubMed2005

The syntrophin-dystrobrevin subcomplex in human neuromuscular disorders.

The syntrophins and alpha-dystrobrevin form a subcomplex with dystrophin at the skeletal muscle membrane, and are also highly concentrated at the neuromuscular synapse. Here we demonstrate that the different syntrophins and alpha-dystrobrevin isoforms have distinct expression patterns during human skeletal muscle development, and are differentially affected by loss of dystrophin anchorage and denervation in human neuromuscular disease. During normal fetal development, and in Duchenne muscular dystrophy and denervation disorders, alpha1-syntrophin and alpha-dystrobrevin are absent or markedly reduced at the sarcolemmal membrane. beta1-Syntrophin is the predominant syntrophin isoform expressed at the muscle membrane during development, and it undergoes upregulation in response to loss of alpha1-syntrophin in Duchenne muscular dystrophy and in denervation. Upregulation of beta1-syntrophin in neuromuscular disorders is associated with re-expression of the fetal nicotinic acetylcholine receptor gamma-subunit, cardiac actin, and neonatal myosin, suggesting reversion of muscle fibers to an immature phenotype. We show that denervation specifically affects expression of the syntrophin-dystrobrevin subcomplex and does not affect levels or localization of other members of the dystrophin-associated protein complex. Our results confirm that dystrophin is required for anchorage of the syntrophin-dystrobrevin subcomplex and suggest that expression of the syntrophin-dystrobrevin complex may be independently regulated through neuromuscular transmission.

Animals↗

PubMed2004

Structural abnormalities at neuromuscular synapses lacking multiple syntrophin isoforms.

The syntrophins are modular adapter proteins that function by recruiting signaling molecules to the cytoskeleton via their direct association with proteins of the dystrophin protein family. We investigated the physiological function of beta2-syntrophin by generating a line of mice lacking this syntrophin isoform. The beta2-syntrophin null mice show no overt phenotype, or muscular dystrophy, and form structurally normal neuromuscular junctions (NMJs). To determine whether physiological consequences caused by the lack of beta2-syntrophin were masked by compensation from the alpha-syntrophin isoform, we crossed these mice with our previously described alpha-syntrophin null mice to produce mice lacking both isoforms. The alpha/beta2-syntrophin null mice have NMJs that are structurally more aberrant than those lacking only alpha-syntrophin. The NMJs of the alpha/beta2-syntrophin null mice have fewer junctional folds than either parent strain, and the remaining folds are abnormally shaped with few openings to the synaptic space. The levels of acetylcholine receptors are reduced to 23% of wild type in mice lacking both syntrophin isoforms. Furthermore, the alpha/beta2-syntrophin null mice ran significantly shorter distances on voluntary exercise wheels despite having normal neuromuscular junction transmission as determined by micro-electrode recording of endplate potentials. We conclude that both alpha-syntrophin and beta2-syntrophin play distinct roles in forming and maintaining NMJ structure and that each syntrophin can partially compensate for the loss of the other.

Animals↗

PubMed2004

Defects in neuromuscular junction structure in dystrophic muscle are corrected by expression of a NOS transgene in dystrophin-deficient muscles, but not in muscles lacking alpha- and beta1-syntrophins.

Muscular dystrophies that arise from mutations of genes that encode proteins in the dystrophin-glycoprotein complex (DGC) frequently involve defects in the structure of neuromuscular junctions (NMJs). DGC mutations that cause NMJ defects typically cause a secondary loss of neuronal nitric oxide synthase (nNOS) from the post-synaptic membrane. We tested the hypothesis that reduction of muscle-derived NO production causes NMJ defects in DGC mutants by analyzing the effect of modulating muscle NO production on NMJ structure in mutant and wild-type muscles. We found that nNOS null mutants, dystrophin-deficient mdx mice and alpha-syntrophin null mutants showed reductions in the concentration of acetylcholine receptors (AChRs) at the post-synaptic membrane. Also, expression of a muscle-specific NOS transgene increased AChR concentration, which reflected an increase in both AChR expression and clustering. NOS transgene expression also increased the size of NMJs, and partially corrected defects in normal NMJ architecture that were observed in mdx and alpha-syntrophin null muscles. In addition, stimulation of AChR clustering in vitro by application of laminin or VVA B4 lectin induced a 3-4-fold increase in NOS activity and increased AChR clustering that could be prevented by NOS inhibition. However, the partial rescue of NMJ structure by expression of a NOS transgene required the expression of alpha- or beta1-syntrophin at the NMJ; partial NMJ rescue was seen in the muscles of alpha-syntrophin mutants that expressed beta1-syntrophin, but no rescue was observed in muscles of alpha-syntrophin mutants that also lacked beta1-syntrophin. These findings show that NO promotes AChR expression and clustering in vivo and contributes to normal NMJ architecture. The results suggest that defects in NMJ structure that occur in some DGC mutants can result from the secondary loss of NOS from muscle.

Animals↗

PubMed2004

The potassium channel Kir4.1 associates with the dystrophin-glycoprotein complex via alpha-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 alpha-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 alpha-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 alpha-syntrophin and suggest an important role for the DGC in central nervous system physiology.

Animals↗

PubMed2004

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 alpha1-, beta1-, and beta2-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.

Amino Acid Motifs↗

PubMed2004

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 alpha-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.

Animals↗

PubMed2003

DAMAGE, a novel alpha-dystrobrevin-associated MAGE protein in dystrophin complexes.

Mice rendered null for alpha-dystrobrevin, a component of the dystrophin complex, have muscular dystrophy, despite the fact that the sarcolemma remains relatively intact (Grady, R. M., Grange, R. W., Lau, K. S., Maimone, M. M., Nichol, M. C., Stull, J. T., and Sanes, J. R. (1999) Nat. Cell Biol. 1, 215-220) Thus, alpha-dystrobrevin may serve a signaling function that is important for the maintenance of muscle integrity. We have identified a new dystrobrevin-associated protein, DAMAGE, that may play a signaling role in brain, muscle, and peripheral nerve. In humans, DAMAGE is encoded by an intronless gene located at chromosome Xq13.1, a locus that contains genes involved in mental retardation. DAMAGE associates directly with alpha-dystrobrevin, as shown by yeast two-hybrid, and co-immunoprecipitates with the dystrobrevin-syntrophin complex from brain. This co-immunoprecipitation is dependent on the presence of alpha-dystrobrevin but not beta-dystrobrevin. The DAMAGE protein contains a potential nuclear localization signal, 30 12-amino acid repeats, and two MAGE homology domains. The domain structure of DAMAGE is similar to that of NRAGE, a MAGE protein that mediates p75 neurotrophin receptor signaling and neuronal apoptosis (Salehi, A. H., Roux, P. P., Kubu, C. J., Zeindler, C., Bhakar, A., Tannis, L. L., Verdi, J. M., and Barker, P. A. (2000) Neuron 27, 279-288). DAMAGE is highly expressed in brain and is present in the cell bodies and dendrites of hippocampal and Purkinje neurons. In skeletal muscle, DAMAGE is at the postsynaptic membrane and is associated with a subset of myonuclei. DAMAGE is also expressed in peripheral nerve, where it localizes along with other members of the dystrophin complex to the perineurium and myelin. These results expand the role of dystrobrevin and the dystrophin complex in membrane signaling and disease.

Amino Acid Sequence↗

PubMed2003

Delayed K+ clearance associated with aquaporin-4 mislocalization: phenotypic defects in brains of alpha-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 alpha-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 alpha-syntrophin-null mice (alpha-Syn-/-). We found that <10% of AQP4 immunogold labeling is retained in the perivascular astrocyte end-feet membranes of the alpha-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 alpha-Syn-/- mice compared with WT mice. Furthermore, the intensity of hyperthermia-induced epileptic seizures was increased in approximately half of the alpha-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.

Animals↗

PubMed2003

Vasomodulation by skeletal muscle-derived nitric oxide requires alpha-syntrophin-mediated sarcolemmal localization of neuronal Nitric oxide synthase.

Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle where it associates with the dystrophin complex at the sarcolemma by binding to the PDZ domain of alpha-syntrophin. Nitric oxide (NO) produced by skeletal muscle nNOS is proposed to regulate blood flow in exercising muscle by diffusing from the skeletal muscle fibers to the nearby microvessels where it attenuates alpha-adrenergic vasoconstriction. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the vasoregulatory effect of skeletal muscle-derived NO. To test this hypothesis, we performed experiments in alpha-syntrophin null mice and in transgenic mice expressing a mutated alpha-syntrophin lacking the PDZ domain (DeltaPDZ), both of which are characterized by reduced sarcolemmal nNOS. We found that modulation of alpha-adrenergic vasoconstriction was greatly impaired in the contracting muscles of the alpha-syntrophin null mice and transgenic DeltaPDZ mice compared with wild-type mice and transgenic mice expressing full-length alpha-syntrophin. These in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the dystrophin-associated protein alpha-syntrophin and may have implications for the development of potential gene therapy strategies to treat muscular dystrophy or other muscle-related diseases.

Animals↗

PubMed2003

An alpha-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 alpha-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 alpha-syntrophin (alpha-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 alpha-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 alpha-Syn(-/-) mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but alpha-syntrophin was retained in perivascular astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus alpha-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 alpha-syntrophin may be a target for treatment of brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule.

Animals↗

PubMed2003

PerleCan fix your muscle AChEs.

Efficient and accurate synaptic transmission requires proper localization of numerous signaling proteins in the synaptic membrane. At the neuromuscular junction, the nicotinic ACh receptor mediates postsynaptic depolarization, and acetylcholinesterase (AChE) terminates this process by hydrolyzing ACh. The mechanism by which the nerve directs receptor localization is understood in considerable detail; AChE clustering, by contrast, has received much less attention. Now, in a recent paper in Nature Neuroscience, the laboratories of Yoshiko Yamada and Richard Rotundo report that AChE clustering at the postsynaptic membrane requires perlecan, which binds both AChE and dystroglycan.

Acetylcholinesterase↗

PubMed2002

GABARAP and GABA(A) receptor clustering.

Coyle et al. (2002), in this issue of Neuron, reveal the crystal structure for the GABA(A) receptor binding protein, GABARAP. They show GABARAP can switch from a monomer to an extended linear polymer form that may function to assemble microtubules during the intracellular trafficking or postsynaptic clustering of GABA(A) receptors.

Adaptor Proteins, Signal Transducing↗