Orchestrating aquaporin-4 and connexin-43 expression in brain: Differential roles of α1- and ß1-syntrophin.

Aquaporin-4 (AQP4) water channels and gap junction proteins (connexins) are two classes of astrocytic membrane proteins critically involved in brain water and ion homeostasis. AQP4 channels are anchored by α1-syntrophin to the perivascular astrocytic endfoot membrane domains where they control water flux at the blood-brain interface while connexins cluster at the lateral aspects of the astrocytic endfeet forming gap junctions that allow water and ions to dissipate through the astrocyte syncytium. Recent studies have pointed to an interdependence between astrocytic AQP4 and astrocytic gap junctions but the underlying mechanism remains to be explored. Here we use a novel transgenic mouse line to unravel whether ß1-syntrophin (coexpressed with α1-syntrophin in astrocytic plasma membranes) is implicated in the expression of AQP4 isoforms and formation of gap junctions in brain. Our results show that while the effect of ß1-syntrophin deletion is rather limited, double knockout of α1- and ß1-syntrophin causes a downregulation of the novel AQP4 isoform AQP4ex and an increase in the number of astrocytic gap junctions. The present study highlight the importance of syntrophins in orchestrating specialized functional domains of brain astrocytes.

Functional specialization of retinal Müller cell endfeet depends on an interplay between two syntrophin isoforms.

Retinal Müller cells are highly polarized macroglial cells with accumulation of the aquaporin-4 (AQP4) water channel and the inwardly rectifying potassium channel Kir4.1 at specialized endfoot membrane domains abutting microvessels and corpus vitreum. Proper water and potassium homeostasis in retina depends on these membrane specializations. Here we show that targeted deletion of ß1-syntrophin leads to a partial loss of AQP4 from perivascular Müller cell endfeet and that a concomitant deletion of both α1- and ß1-syntrophin causes a near complete loss of AQP4 from both perivascular and subvitreal endfoot membranes. α1-syntrophin is normally very weakly expressed in Müller cell endfeet but ß1-syntrophin knockout mice display an increased amount of α1-syntrophin at these sites. We suggest that upregulation of perivascular α1-syntrophin restricts the effect of ß1-syntrophin deletion. The present findings indicate that ß1-syntrophin plays an important role in maintaining the functional polarity of Müller cells and that α1-syntrophin can partially substitute for ß1-syntrophin in AQP4 anchoring. Functional polarization of Müller cells thus depends on an interplay between two syntrophin isoforms.

Simvastatin provides long-term improvement of left ventricular function and prevents cardiac fibrosis in muscular dystrophy.

Duchenne muscular dystrophy (DMD), caused by absence of the protein dystrophin, is a common, degenerative muscle disease affecting 1:5000 males worldwide. With recent advances in respiratory care, cardiac dysfunction now accounts for 50% of mortality in DMD. Recently, we demonstrated that simvastatin substantially improved skeletal muscle health and function in mdx (DMD) mice. Given the known cardiovascular benefits ascribed to statins, the aim of this study was to evaluate the efficacy of simvastatin on cardiac function in mdx mice. Remarkably, in 12-month old mdx mice, simvastatin reversed diastolic dysfunction to normal after short-term treatment (8 weeks), as measured by echocardiography in animals anesthetized with isoflurane and administered dobutamine to maintain a physiological heart rate. This improvement in diastolic function was accompanied by increased phospholamban phosphorylation in simvastatin-treated mice. Echocardiography measurements during long-term treatment, from 6 months up to 18 months of age, showed that simvastatin significantly improved in vivo cardiac function compared to untreated mdx mice, and prevented fibrosis in these very old animals. Cardiac dysfunction in DMD is also characterized by decreased heart rate variability (HRV), which indicates autonomic function dysregulation. Therefore, we measured cardiac ECG and demonstrated that short-term simvastatin treatment significantly increased heart rate variability (HRV) in 14-month-old conscious mdx mice, which was reversed by atropine. This finding suggests that enhanced parasympathetic function is likely responsible for the improved HRV mediated by simvastatin. Together, these findings indicate that simvastatin markedly improves cardiac health and function in dystrophic mice, and therefore may provide a novel approach for treating cardiomyopathy in DMD.

Targeted deletion of ß1-syntrophin causes a loss of Kir 4.1 from Müller cell endfeet in mouse retina.

Proper function of the retina depends heavily on a specialized form of retinal glia called Müller cells. These cells carry out important homeostatic functions that are contingent on their polarized nature. Specifically, the Müller cell endfeet that contact retinal microvessels and the corpus vitreum show a tenfold higher concentration of the inwardly rectifying potassium channel Kir 4.1 than other Müller cell plasma membrane domains. This highly selective enrichment of Kir 4.1 allows K+ to be siphoned through endfoot membranes in a special form of spatial buffering. Here, we show that Kir 4.1 is enriched in endfoot membranes through an interaction with ß1-syntrophin. Targeted disruption of this syntrophin caused a loss of Kir 4.1 from Müller cell endfeet without affecting the total level of Kir 4.1 expression in the retina. Targeted disruption of α1-syntrophin had no effect on Kir 4.1 localization. Our findings show that the Kir 4.1 aggregation that forms the basis for K+ siphoning depends on a specific syntrophin isoform that colocalizes with Kir 4.1 in Müller endfoot membranes.

Mice lacking α-, ß1- and ß2-syntrophins exhibit diminished function and reduced dystrophin expression in both cardiac and skeletal muscle.

Syntrophins are a family of modular adaptor proteins that are part of the dystrophin protein complex, where they recruit and anchor a variety of signaling proteins. Previously we generated mice lacking α- and/or ß2-syntrophin but showed that in the absence of one isoform, other syntrophin isoforms can partially compensate. Therefore, in the current study, we generated mice that lacked α, ß1 and ß2-syntrophins [triple syntrophin knockout (tKO) mice] and assessed skeletal and cardiac muscle function. The tKO mice showed a profound reduction in voluntary wheel running activity at both 6 and 12 months of age. Function of the tibialis anterior was assessed in situ and we found that the specific force of tKO muscle was decreased by 20-25% compared with wild-type mice. This decrease was accompanied by a shift in fiber-type composition from fast 2B to more oxidative fast 2A fibers. Using echocardiography to measure cardiac function, it was revealed that tKO hearts had left ventricular cardiac dysfunction and were hypertrophic, with a thicker left ventricular posterior wall. Interestingly, we also found that membrane-localized dystrophin expression was lower in both skeletal and cardiac muscles of tKO mice. Since dystrophin mRNA levels were not different in tKO, this finding suggests that syntrophins may regulate dystrophin trafficking to, or stabilization at, the sarcolemma. These results show that the loss of all three major muscle syntrophins has a profound effect on exercise performance, and skeletal and cardiac muscle dysfunction contributes to this deficiency.

Syntrophin binds directly to multiple spectrin-like repeats in dystrophin and mediates binding of nNOS to repeats 16-17.

Mutation of the gene encoding dystrophin leads to Duchenne and Becker muscular dystrophy (DMD and BMD). Currently, dystrophin is thought to function primarily as a structural protein, connecting the muscle cell actin cytoskeleton to the extra-cellular matrix. In addition to this structural role, dystrophin also plays an important role as a scaffold that organizes an array of signaling proteins including sodium, potassium, and calcium channels, kinases, and nitric oxide synthase (nNOS). Many of these signaling proteins are linked to dystrophin via syntrophin, an adapter protein that is known to bind directly to two sites in the carboxyl terminal region of dystrophin. A search of the dystrophin sequence revealed three additional potential syntrophin binding sites (SBSs) within the spectrin-like repeat (SLR) region of dystrophin. Binding assays revealed that the site at SLR 17 bound specifically to the α isoform of syntrophin while the site at SLR 22 bound specifically to the ß-syntrophins. The SLR 17 α-SBS contained the core sequence known to be required for nNOS-dystrophin interaction. In vitro and in vivo assays indicate that α-syntrophin facilitates the nNOS-dystrophin interaction at this site rather than nNOS binding directly to dystrophin as previously reported. The identification of multiple SBSs within the SLR region of dystrophin demonstrates that this region functions as a signaling scaffold. The signaling role of the SLR region of dystrophin will need to be considered for effective gene replacement or exon skipping based DMD/BMD therapies.

Validation of ultrasonography for non-invasive assessment of diaphragm function in muscular dystrophy.

KEY POINTS: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease that is commonly studied using the mdx mouse. The mdx diaphragm muscle closely mimics the pathophysiological changes in DMD muscles. mdx diaphragm force is commonly assessed ex vivo, precluding time course studies. Here we used ultrasonography to evaluate time-dependent changes in diaphragm function in vivo, by measuring diaphragm movement amplitude. In mdx mice, diaphragm amplitude decreased with age and values were much lower than for wild-type mice. Importantly, diaphragm amplitude strongly correlated with ex vivo specific force values. Micro-dystrophin administration increased mdx diaphragm amplitude by 26% after 4 weeks. Diaphragm amplitude correlated positively with ex vivo force values and negatively with diaphragm fibrosis, a major cause of DMD muscle weakness. These studies validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in mdx diaphragm function in vivo. This technique will be valuable for testing potential therapies for DMD. ABSTRACT: Duchenne muscular dystrophy (DMD) is a severe, degenerative muscle disease caused by dystrophin mutations. The mdx mouse is a widely used animal model of DMD. The mdx diaphragm muscle most closely recapitulates key features of DMD muscles, including progressive fibrosis and considerable force loss. Diaphragm function in mdx mice is commonly evaluated by specific force measurements ex vivo. While useful, this method only measures force from a small muscle sample at one time point. Therefore, accurate assessment of diaphragm function in vivo would provide an important advance to study the time course of functional decline and treatment benefits. Here, we evaluated an ultrasonography technique for measuring time-dependent changes of diaphragm function in mdx mice. Diaphragm movement amplitude values for mdx mice were considerably lower than those for wild-type, decreased from 8 to 18 months of age, and correlated strongly with ex vivo specific force. We then investigated the time course of diaphragm amplitude changes following administration of an adeno-associated viral vector expressing Flag-micro-dystrophin (AAV-µDys) to young adult mdx mice. Diaphragm amplitude peaked 4 weeks after AAV-µDys administration, and was 26% greater than control mdx mice at this time. This value decreased slightly to 21% above mdx controls after 12 weeks of treatment. Importantly, diaphragm amplitude again correlated strongly with ex vivo specific force. Also, diaphragm amplitude and specific force negatively correlated with fibrosis levels in the muscle. Together, our results validate diaphragm ultrasonography as a reliable technique for assessing time-dependent changes in dystrophic diaphragm function in vivo, and for evaluating potential therapies for DMD.

Simvastatin offers new prospects for the treatment of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common and severe inherited neuromuscular disorder. DMD is caused by mutations in the gene encoding the dystrophin protein in muscle fibers. Dystrophin was originally proposed to be a structural protein that protected the sarcolemma from stresses produced during contractions. However, more recently, experimental evidence has revealed a far more complicated picture, with the loss of dystrophin causing dysfunction of multiple muscle signaling pathways, which all contribute to the overall disease pathophysiology. Current gene-based approaches for DMD are conceptually appealing since they offer the potential to restore dystrophin to muscles, albeit a partially functional, truncated form of the protein. However, given the cost and technical challenges facing these genetic approaches, it is important to consider if relatively inexpensive, clinically used drugs may be repurposed for treating DMD. Here, we discuss our recent findings showing the potential of simvastatin as a novel therapy for DMD.

Altered short-term synaptic plasticity and reduced muscle strength in mice with impaired regulation of presynaptic CaV2.1 Ca2+ channels.

Facilitation and inactivation of P/Q-type calcium (Ca(2+)) currents through the regulation of voltage-gated Ca(2+) (CaV) 2.1 channels by Ca(2+) sensor (CaS) proteins contributes to the facilitation and rapid depression of synaptic transmission in cultured neurons that transiently express CaV2.1 channels. To examine the modulation of endogenous CaV2.1 channels by CaS proteins in native synapses, we introduced a mutation (IM-AA) into the CaS protein-binding site in the C-terminal domain of CaV2.1 channels in mice, and tested synaptic facilitation and depression in neuromuscular junction synapses that use exclusively CaV2.1 channels for Ca(2+) entry that triggers synaptic transmission. Even though basal synaptic transmission was unaltered in the neuromuscular synapses in IM-AA mice, we found reduced short-term facilitation in response to paired stimuli at short interstimulus intervals in IM-AA synapses. In response to trains of action potentials, we found increased facilitation at lower frequencies (10-30 Hz) in IM-AA synapses accompanied by slowed synaptic depression, whereas synaptic facilitation was reduced at high stimulus frequencies (50-100 Hz) that would induce strong muscle contraction. As a consequence of altered regulation of CaV2.1 channels, the hindlimb tibialis anterior muscle in IM-AA mice exhibited reduced peak force in response to 50 Hz stimulation and increased muscle fatigue. The IM-AA mice also had impaired motor control, exercise capacity, and grip strength. Taken together, our results indicate that regulation of CaV2.1 channels by CaS proteins is essential for normal synaptic plasticity at the neuromuscular junction and for muscle strength, endurance, and motor coordination in mice in vivo.

Sarcolemmal targeting of nNOSµ improves contractile function of mdx muscle.

Nitric oxide (NO) is a key regulator of skeletal muscle function and metabolism, including vasoregulation, mitochondrial function, glucose uptake, fatigue and excitation-contraction coupling. The main generator of NO in skeletal muscle is the muscle-specific form of neuronal nitric oxide synthase (nNOSµ) produced by the NOS1 gene. Skeletal muscle nNOSµ is predominantly localized at the sarcolemma by interaction with the dystrophin protein complex (DPC). In Duchenne muscular dystrophy (DMD), loss of dystrophin leads to the mislocalization of nNOSµ from the sarcolemma to the cytosol. This perturbation has been shown to impair contractile function and cause muscle fatigue in dystrophic (mdx) mice. Here, we investigated the effect of restoring sarcolemmal nNOSµ on muscle contractile function in mdx mice. To achieve this, we designed a modified form of nNOSµ (NOS-M) that is targeted to the sarcolemma by palmitoylation, even in the absence of the DPC. When expressed specifically in mdx skeletal muscle, NOS-M significantly attenuates force loss owing to damaging eccentric contractions and repetitive isometric contractions (fatigue), while also improving force recovery after fatigue. Expression of unmodified nNOSµ at similar levels does not lead to sarcolemmal association and fails to improve muscle function. Aside from the benefits of sarcolemmal-localized NO production, NOS-M also increased the surface membrane levels of utrophin and other DPC proteins, including ß-dystroglycan, α-syntrophin and α-dystrobrevin in mdx muscle. These results suggest that the expression of NOS-M in skeletal muscle may be therapeutically beneficial in DMD and other muscle diseases characterized by the loss of nNOSµ from the sarcolemma.

Absence of Dystrophin Disrupts Skeletal Muscle Signaling: Roles of Ca2+, Reactive Oxygen Species, and Nitric Oxide in the Development of Muscular Dystrophy.

Dystrophin is a long rod-shaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC components cause the muscular dystrophies, a group of inherited diseases in which repeated bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The normal function of dystrophin is poorly defined. In its absence a complex series of changes occur with multiple muscle proteins showing reduced or increased expression or being modified in various ways. In this review, we will consider the various proteins whose expression and function is changed in muscular dystrophies, focusing on Ca(2+)-permeable channels, nitric oxide synthase, NADPH oxidase, and caveolins. Excessive Ca(2+) entry, increased membrane permeability, disordered caveolar function, and increased levels of reactive oxygen species are early changes in the disease, and the hypotheses for these phenomena will be critically considered. The aim of the review is to define the early damage pathways in muscular dystrophy which might be appropriate targets for therapy designed to minimize the muscle degeneration and slow the progression of the disease.

A new therapeutic effect of simvastatin revealed by functional improvement in muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal, degenerative muscle disease with no effective treatment. DMD muscle pathogenesis is characterized by chronic inflammation, oxidative stress, and fibrosis. Statins, cholesterol-lowering drugs, inhibit these deleterious processes in ischemic diseases affecting skeletal muscle, and therefore have potential to improve DMD. However, statins have not been considered for DMD, or other muscular dystrophies, principally because skeletal-muscle-related symptoms are rare, but widely publicized, side effects of these drugs. Here we show positive effects of statins in dystrophic skeletal muscle. Simvastatin dramatically reduced damage and enhanced muscle function in dystrophic (mdx) mice. Long-term simvastatin treatment vastly improved overall muscle health in mdx mice, reducing plasma creatine kinase activity, an established measure of muscle damage, to near-normal levels. This reduction was accompanied by reduced inflammation, more oxidative muscle fibers, and improved strength of the weak diaphragm muscle. Shorter-term treatment protected against muscle fatigue and increased mdx hindlimb muscle force by 40%, a value comparable to current dystrophin gene-based therapies. Increased force correlated with reduced NADPH Oxidase 2 protein expression, the major source of oxidative stress in dystrophic muscle. Finally, in old mdx mice with severe muscle degeneration, simvastatin enhanced diaphragm force and halved fibrosis, a major cause of functional decline in DMD. These improvements were accompanied by autophagy activation, a recent therapeutic target for DMD, and less oxidative stress. Together, our findings highlight that simvastatin substantially improves the overall health and function of dystrophic skeletal muscles and may provide an unexpected, novel therapy for DMD and related neuromuscular diseases.

Evaluation of the specificity of four commercially available antibodies to alpha-syntrophin.

Alpha-syntrophin (SNTA) is an adaptor protein that regulates several signaling pathways. To analyze expression of SNTA immunoblot assays must be performed. Here, the specificity of four commercially available SNTA antibodies has been evaluated in immunoblot experiments using liver tissues of wild-type and SNTA-deficient mice. While one of the antibodies reacts with SNTA, two antibodies specifically recognize beta 2 syntrophin (SNTB2). The antigen detected by the fourth antibody has not been identified but is different from SNTA and SNTB2. Therefore, only one of the four tested antibodies is appropriate to analyze SNTA protein levels by immunoblot.

Lipid abnormalities in alpha/beta2-syntrophin null mice are independent from ABCA1.

The syntrophins alpha (SNTA) and beta 2 (SNTB2) are molecular adaptor proteins shown to stabilize ABCA1, an essential regulator of HDL cholesterol. Furthermore, SNTB2 is involved in glucose stimulated insulin release. Hyperglycemia and dyslipidemia are characteristic features of the metabolic syndrome, a serious public health problem with rising prevalence. Therefore, it is important to understand the role of the syntrophins herein. Mice deficient for both syntrophins (SNTA/B2-/-) have normal insulin and glucose tolerance, hepatic ABCA1 protein and cholesterol. When challenged with a HFD, wild type and SNTA/B2-/- mice have similar weight gain, adiposity, serum and liver triglycerides. Hepatic ABCA1, serum insulin and insulin sensitivity are normal while glucose tolerance is impaired. Liver cholesterol is reduced, and expression of SREBP2 and HMG-CoA-R is increased in the knockout mice. Scavenger receptor-BI (SR-BI) protein is strongly diminished in the liver of SNTA/B2-/- mice while SR-BI binding protein NHERF1 is not changed and PDZK1 is even induced. Knock-down of SNTA, SNTB2 or both has no effect on hepatocyte SR-BI and PDZK1 proteins. Further, SR-BI levels are not reduced in brown adipose tissue of SNTA/B2-/- mice excluding that syntrophins directly stabilize SR-BI. SR-BI stability is regulated by MAPK and phosphorylated ERK2 is induced in the liver of the knock-out mice. Blockage of ERK activity upregulates hepatocyte SR-BI showing that increased MAPK activity contributes to low SR-BI. Sphingomyelin which is well described to regulate cholesterol metabolism is reduced in the liver and serum of the knock-out mice while the size of serum lipoproteins is not affected. Current data exclude a major function of these syntrophins in ABCA1 activity and insulin release but suggest a role in regulating glucose uptake, ERK and SR-BI levels, and sphingomyelin metabolism in obesity.

Loss of nNOS inhibits compensatory muscle hypertrophy and exacerbates inflammation and eccentric contraction-induced damage in mdx mice.

Approaches targeting nitric oxide (NO) signaling show promise as therapies for Duchenne and Becker muscular dystrophies. However, the mechanisms by which NO benefits dystrophin-deficient muscle remain unclear, but may involve nNOSß, a newly discovered enzymatic source of NO in skeletal muscle. Here we investigate the impact of dystrophin deficiency on nNOSß and use mdx mice engineered to lack nNOSµ and nNOSß to discern how the loss of nNOS impacts dystrophic skeletal muscle pathology. In mdx muscle, nNOSß was mislocalized and its association with the Golgi complex was reduced. nNOS depletion from mdx mice prevented compensatory skeletal muscle cell hypertrophy, decreased myofiber central nucleation and increased focal macrophage cell infiltration, indicating exacerbated dystrophic muscle damage. Reductions in muscle integrity in nNOS-null mdx mice were accompanied by decreases in specific force and increased susceptibility to eccentric contraction-induced muscle damage compared with mdx controls. Unexpectedly, muscle fatigue was unaffected by nNOS depletion, revealing a novel latent compensatory mechanism for the loss of nNOS in mdx mice. Together with previous studies, these data suggest that localization of both nNOSµ and nNOSß is disrupted by dystrophin deficiency. They also indicate that nNOS has a more complex role as a modifier of dystrophic pathology and broader therapeutic potential than previously recognized. Importantly, these findings also suggest nNOSß as a new drug target and provide a new conceptual framework for understanding nNOS signaling and the benefits of NO therapies in dystrophinopathies.

Adiponectin receptor 1 C-terminus interacts with PDZ-domain proteins such as syntrophins.

Adiponectin receptor 1 (AdipoR1) is one of the two signaling receptors of adiponectin with multiple beneficial effects in metabolic diseases. AdipoR1 C-terminal peptide is concordant with the consensus sequence of class I PSD-95, disc large, ZO-1 (PDZ) proteins, and screening of a liver yeast two hybrid library identified binding to ß2-syntrophin (SNTB2). Hybridization of a PDZ-domain array with AdipoR1 C-terminal peptide shows association with PDZ-domains of further proteins including ß1- and α-syntrophin (SNTA). Interaction of PDZ proteins and C-terminal peptides requires a free carboxy terminus next to the PDZ-binding region and is blocked by carboxy terminal added tags. N-terminal tagged AdipoR1 is more highly expressed than C-terminal tagged receptor suggesting that the free carboxy terminus may form a complex with PDZ proteins to regulate cellular AdipoR1 levels. The C- and N-terminal tagged AdipoR1 proteins are mainly localized in the cytoplasma. N-terminal but not C-terminal tagged AdipoR1 colocalizes with syntrophins in adiponectin incubated Huh7 cells. Adiponectin induced hepatic phosphorylation of AMPK and p38 MAPK which are targets of AdipoR1 is, however, not blocked in SNTA and SNTB2 deficient mice. Further, AdipoR1 protein is similarly abundant in the liver of knock-out and wild type mice when kept on a standard chow or a high fat diet. In summary these data suggest that AdipoR1 protein levels are regulated by so far uncharacterized class I PDZ proteins which are distinct from SNTA and SNTB2.

Regional registration of [6-(14)C]glucose metabolism during brain activation of α-syntrophin knockout mice.

α-Syntrophin is a component of the dystrophin scaffold-protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α-Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K(+) homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4-mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [(14)C]metabolites during sensory stimulation. Conscious KO and wild-type mice were pulse-labeled with [6-(14)C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer-assisted brain imaging or analysis of [(14)C]metabolites in extracts, respectively. High-resolution autoradiographic assays detected a 17% side-to-side difference (p < 0.05) in inferior colliculus of KO mice, not wild-type mice. However, there were no labeling differences between KO and wild-type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4-mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.

The light chains of microtubule-associated proteins MAP1A and MAP1B interact with α1-syntrophin in the central and peripheral nervous system.

Microtubule-associated proteins of the MAP1 family (MAP1A, MAP1B, and MAP1S) share, among other features, a highly conserved COOH-terminal domain approximately 125 amino acids in length. We conducted a yeast 2-hybrid screen to search for proteins interacting with this domain and identified α1-syntrophin, a member of a multigene family of adapter proteins involved in signal transduction. We further demonstrate that the interaction between the conserved COOH-terminal 125-amino acid domain (which is located in the light chains of MAP1A, MAP1B, and MAP1S) and α1-syntrophin is direct and occurs through the pleckstrin homology domain 2 (PH2) and the postsynaptic density protein 95/disk large/zonula occludens-1 protein homology domain (PDZ) of α1-syntrophin. We confirmed the interaction of MAP1B and α1-syntrophin by co-localization of the two proteins in transfected cells and by co-immunoprecipitation experiments from mouse brain. In addition, we show that MAP1B and α1-syntrophin partially co-localize in Schwann cells of the murine sciatic nerve during postnatal development and in the adult. However, intracellular localization of α1-syntrophin and other Schwann cell proteins such as ezrin and dystrophin-related protein 2 (DRP2) and the localization of the axonal node of Ranvier-associated protein Caspr1/paranodin were not affected in MAP1B null mice. Our findings add to a growing body of evidence that classical MAPs are likely to be involved in signal transduction not only by directly modulating microtubule function, but also through their interaction with signal transduction proteins.

Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins.

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality. Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac-specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in Duchenne and Becker muscular dystrophies.

Sildenafil reduces respiratory muscle weakness and fibrosis in the mdx mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy caused by mutations in the dystrophin gene. Loss of dystrophin initiates a progressive decline in skeletal muscle integrity and contractile capacity which weakens respiratory muscles including the diaphragm, culminating in respiratory failure, the leading cause of morbidity and mortality in DMD patients. At present, corticosteroid treatment is the primary pharmacological intervention in DMD, but has limited efficacy and adverse side effects. Thus, there is an urgent need for new safe, cost-effective, and rapidly implementable treatments that slow disease progression. One promising new approach is the amplification of nitric oxide-cyclic guanosine monophosphate (NO-cGMP) signalling pathways with phosphodiesterase 5 (PDE5) inhibitors. PDE5 inhibitors serve to amplify NO signalling that is attenuated in many neuromuscular diseases including DMD. We report here that a 14-week treatment of the mdx mouse model of DMD with the PDE5 inhibitor sildenafil (Viagra(®), Revatio(®)) significantly reduced mdx diaphragm muscle weakness without impacting fatigue resistance. In addition to enhancing respiratory muscle contractility, sildenafil also promoted normal extracellular matrix organization. PDE5 inhibition slowed the establishment of mdx diaphragm fibrosis and reduced matrix metalloproteinase-13 (MMP-13) expression. Sildenafil also normalized the expression of the pro-fibrotic (and pro-inflammatory) cytokine tumour necrosis factor α (TNFα). Sildenafil-treated mdx diaphragms accumulated significantly less Evans Blue tracer dye than untreated controls, which is also indicative of improved diaphragm muscle health. We conclude that sildenafil-mediated PDE5 inhibition significantly reduces diaphragm respiratory muscle dysfunction and pathology in the mdx mouse model of Duchenne muscular dystrophy. This study provides new insights into the therapeutic utility of targeting defects in NO-cGMP signalling with PDE5 inhibitors in dystrophin-deficient muscle.