The domain of brain beta-spectrin responsible for synaptic vesicle association is essential for synaptic transmission

We have examined the interaction between synapsin I, the major phosphoprotein on the membrane of small synaptic vesicles, and brain spectrin. Using recombinant peptides we have localized the synapsin I attachment site upon the beta-spectrin isoform betaSpIISigmaI to a region of 25 amino acids, residues 211 through 235. This segment is adjacent to the actin binding domain and is within the region of the betaSpIISigmaI that we previously predicted as a candidate synapsin I binding domain based upon sequence homology. We used differential centrifugation techniques to quantitatively assess the interaction of spectrin with synaptic vesicles. Using this assay, high affinity saturable binding of recombinant betaSpIISigmaI proteins was observed with synaptic vesicles. Binding was only observed when the 25 amino acid synapsin I binding site was included on the recombinant peptides. Further, we demonstrate that antibodies directed against 15 amino acids of the synapsin I binding domain specifically blocked synaptic transmission in cultured hippocampal neurons. Thus, the synapsin I attachment site on betaSpIISigmaI spectrin comprises a approximately 25 amino acid segment of the molecule and interaction of these two proteins is an essential step for the process of neurotransmission.     

Spectrin (βSpIIΣ1) is an essential component of synaptic transmission

The cellular mechanism that underlies the regulated release of synaptic vesicles during neurotransmission is not fully known. Our previous data has shown that brain spectrin (αSpIIΣ1/βSpIIΣ1)₂ is localized in axons and nerve terminals and we have shown that the β subunit (βSpIIΣ1) contains a synapsin-binding domain capable of interacting with synapsin and small synaptic vesicles in vitro and in vivo. These findings suggested a role for brain β-spectrin in synaptic neurotransmission. To examine this possibility further, peptide-specific antibodies directed against epitopes within the synapsin-binding domain of brain β-spectrin, or against flanking regions, were injected into the presynaptic neuron of synaptically paired rat hippocampal neurons in culture. Here, we show that the antibodies directed against the synapsin-binding domain specifically blocked synaptic neurotransmission.     

The effect of synapsin I phosphorylation upon binding of synaptic vesicles to spectrin.

We have previously demonstrated that brain spectrin is attached to small spherical synaptic vesicles via synapsin I. These studies utilized a novel microfiltration assay in which 125I-labelled synaptic vesicles were incubated with brain spectrin which was covalently attached to cellulosic membranes. In these studies purified dephosphosynapsin I was demonstrated to competitively inhibit the binding of the synaptic vesicles to the immobilized brain spectrin with a KI = 45 nM. In the current study we demonstrate that phosphorylation of synapsin I site 1 (0.74 mol Pi/mol synapsin I) with cAMP-dependent protein kinase and sites 2 and 3 (2.0 mol Pi/mol synapsin I) with Ca(2+)-calmodulin kinase II had little effect upon its interaction with brain spectrin. cAMP-dependent protein kinase phosphorylated synapsin I and Ca(2+)-calmodulin kinase II phosphorylated synapsin I both inhibited the binding of 125I-labelled synaptic vesicles to immobilized brain spectrin with a KI of 23 nM and 24 nM respectively. We conclude that phosphorylation of synapsin I does not down-regulate the interaction of synaptic vesicles with brain spectrin.     

Amelin and synapsin I are 4.1 related spectrin binding proteins in brain

How do synaptic vesicles move towards the presynaptic plasma membrane, fuse with that membrane, and release their contents during synaptic transmission? The answers to these questions at the molecular level are just beginning to be understood. Synapsin I is a neuron specific phosphoprotein that is associated with the cytoplasmic surface of synaptic vesicles. During synaptic transmission, the translocation of the synaptic vesicles to the presynaptic membrane of the neuron is thought to be mediated through changes in the phosphorylation state of synapsin I. It has been suggested that synapsin I is a spectrin binding protein related to the erythrocyte cytoskeletal protein 4.1, which binds to the terminal ends of the erythrocyte spectrin tetramer. The interaction of synapsin I (through brain spectrin) with the neuronal cytoskeleton may be essential for regulating the movement of synaptic vesicles towards the presynaptic plasma membrane. In addition, we have identified another protein in brain that is immunologically and structurally more closely related to erythrocyte 4.1 than is synapsin I. This protein, termed amelin, is localized in the cell body and dendrites of the neuron, whereas synapsin I is found exclusively in the synaptic terminals, suggesting that there is a family of erythrocyte 4.1 related proteins present in brain with distinct subcellular distribution and functions.     

Synapsin I: A regulated synaptic vesicle organizing protein

Synapsin is a protein initially discovered and characterized as a target for cyclic AMP and Ca/calmodulin-regulated protein kinases that is concentrated in nerve endings and is localized on the surface of small synaptic vesicles. Synapsin shares antigenic sites and some local regions of homology with erythrocyte protein 4.1, although these proteins in general are quite different in sequence. Protein 4.1 and synapsin share several local regions of homology with erythrocyte spectrin alpha subunit. Protein 4.1 and synapsin may be related to each other through a common relationship with spectrin. Synapsin binds to synaptic vesicles and membrane sites with a Kd of 0.01-0.02 microM and associates with a Kd of 0.5-4 microM to spectrin, microtubules and neurofilaments in in vitro assays. Synapsin interconnects synaptic vesicles to membranes, and this activity is inhibited by phosphorylation with Ca/calmodulin-dependent protein kinase. Synapsin may have a role in neurons as a structural protein capable of interconnecting small synaptic vesicles with a number of proteins, including spectrin, microtubules, neurofilaments, and membrane sites. A physiological function of synapsin could be as a vesicle-organizing protein that mediates calcium-regulated association of vesicles with cytoskeletal proteins during axonal transport and attaches vesicles to active zones in nerve endings.     

Tissue-type plasminogen activator triggers the synaptic vesicle cycle in cerebral cortical neurons

The active zone (AZ) is a thickening of the presynaptic membrane where exocytosis takes place. Chemical synapses contain neurotransmitter-loaded synaptic vesicles (SVs) that at rest are tethered away from the synaptic release site, but after the presynaptic inflow of Ca⁺² elicited by an action potential translocate to the AZ to release their neurotransmitter load. We report that tissue-type plasminogen activator (tPA) is stored outside the AZ of cerebral cortical neurons, either intermixed with small clear-core vesicles or in direct contact with the presynaptic membrane. We found that cerebral ischemia-induced release of neuronal tPA, or treatment with recombinant tPA, recruits the cytoskeletal protein βII-spectrin to the AZ and promotes the binding of SVs to βII-spectrin, enlarging the population of SVs in proximity to the synaptic release site. This effect does not require the generation of plasmin and is followed by the recruitment of voltage gated calcium channels (VGCC) to the presynaptic terminal that leads to Ca⁺²-dependent synapsin I phosphorylation, freeing SVs to translocate to the AZ to deliver their neurotransmitter load. Our studies indicate that tPA activates the SV cycle and induces the structural and functional changes in the synapse that are required for successful neurotransmission.     

Spectrin (betaSpIIsigma1) is an essential component of synaptic transmission

The cellular mechanism that underlies the regulated release of synaptic vesicles during neurotransmission is not fully known. Our previous data has shown that brain spectrin (alphaSpIIsigma1/betaSpIIsigma1)2 is localized in axons and nerve terminals and we have shown that the beta subunit (betaSpIIsigma1) contains a synapsin-binding domain capable of interacting with synapsin and small synaptic vesicles in vitro and in vivo. These findings suggested a role for brain beta-spectrin in synaptic neurotransmission. To examine this possibility further, peptide-specific antibodies directed against epitopes within the synapsin-binding domain of brain beta-spectrin, or against flanking regions, were injected into the presynaptic neuron of synaptically paired rat hippocampal neurons in culture. Here, we show that the antibodies directed against the synapsin-binding domain specifically blocked synaptic neurotransmission.     

α-spectrins are major ubiquitinated proteins in rat hippocampal neurons and components of ubiquitinated inclusions in neurodegenerative disorders

We have demonstrated that α-spectrins (αSpIΣ* and αSpIIΣ1) are major ubiquitinated proteins in terminally differentiated hippocampal neurons in culture. Western blotting experiments, using αSpIΣ1, αSpIIΣ1, and ubiquitin antibodies and lysates of 11-day-old cultured rat hippocampal neurons, have demonstrated that a single band comigrating with αSpIΣ* and αSpIIΣ1 in a 5% polyacrylamide sodium dodecyl sulfate gel is recognized by ubiquitin antibodies when ¹²⁵I-protein A is used for detection. Immunofluorescence staining of the 7- and 12 -day-old rat hippocampal neuron cultures using ubiquitin, αSpIΣ1, and αSpIIΣ1 antibodies demonstrated that all of these antibodies label neurons but not the astrocytes in the cultures. Immunoprecipitation of spectrin subunits in lysates of 12-day-old rat hippocampal neurons under stringent conditions (9.5 M urea) using αSpIΣ1 and αSpIIΣ1 antibodies followed by Western blot experiments of the immunoprecipitated spectrin subunits using αSpIΣ1, αSpIIΣ1 and ubiquitin antibodies confirmed that both αSpIΣ* and αSpIIΣ1 are ubiquitinated in rat hippocampal neurons. Furthermore, we demonstrated by immunohistochemistry that α-spectrins are components of the cytoplasmic ubiquitinated inclusions in hippocampal neurons in Alzheimer’s and Parkinson’s disease patients.     

Axonal transport of synapsin I-like proteins in rabbit retinal ganglion cells

Synapsin I is a neuronal phosphoprotein that is associated with the cytoplasmic surface of small, clear synaptic vesicles in neuronal synaptic terminals; it may play an important role in synaptic transmission. In vitro, it can interact with fodrin, a relative of the erythrocyte protein spectrin. We have investigated the delivery of synapsin I from its site of synthesis in neuronal cell bodies to synaptic terminals by means of the process of axonal transport. We labeled the newly synthesized proteins of rabbit retinal ganglion cells by injecting 35S-methionine into the vitreous humour, and subsequently observed the appearance of radioactive synapsin I (identified by its 2-dimensional electrophoretic mobility) in tissues containing the axons and synaptic terminals of these neurons. A portion of the newly synthesized synapsin I was axonally transported at the velocity of the most rapidly transported (group I) proteins, which comprise membrane-associated proteins and may include elements of synaptic vesicles. However, the subsequent time course of labeling of synapsin I in the axons suggests that greater than 90% of the axonally transported synapsin I may comprise 2 additional populations--one transported rapidly, the other slowly--that are released from the cell bodies only after a delay of more than 1 d. The delayed, slowly transported population moves at the velocity (approximately 6 mm/d) of groups III and IV (which include fodrin and other proteins of the membrane cytoskeleton). We consider whether such distinct populations may correspond to functionally specialized variants of synapsin I-like proteins that may be transported in association with different organelles. The electrophoretic mobility of labeled synapsin I-like proteins in the axons changed subtly with time. Additional subtle differences between labeled synapsin I-like proteins in the axons and the terminal-containing tissues suggest that certain posttranslational modifications occur specifically in the terminals.     

Abnormality of synaptic vesicular associated proteins in cerebral cortex and hippocampus after microwave exposure

Studies were performed to determine the effects of microwave on synaptic vesicles and the expression of synaptic vesicular associated proteins including synapsin I, VAMP‐2, syntaxin, and synaptophysin. 25 Wistar rats were exposed to microwave which the average power density was 30 mW/cm², and whole body average specific absorption rate was 14.1 W/kg for 5 min. Synaptosome preparations in the cerebral cortex and hippocampus were obtained by isotonic Percoll/sucrose discontinuous gradients at 6 h, 1, 3, and 7 days after radiation. The expression of synaptic vesicular associated proteins was measured using Western blots and image analysis. The interaction between VAMP‐2 and syntaxin was examined by coimmunoprecipitation analysis. Synapsin I in the cerebral cortex were decreased at 3 days (P < 0.01) after radiation and in the hippocampus increased at 1 day (P < 0.01), decreased at 3 days (P < 0.01), increased again at 7 days (P < 0.01) after exposure, compared with the sham‐treated controls. Synaptophysin were increased in 1–7 days (P < 0.01) after exposure in the cerebral cortex and hippocampus. VAMP‐2 were decreased at 1 and 3 days (P < 0.01) and syntaxin were decreased in 6 h to 3 days (P < 0.01) after radiation in the cerebral cortex and hippocampus. The interactions between VAMP‐2 and syntaxin were decreased at 3–7 days (P < 0.01) after radiation in the cerebral cortex and hippocampus, compared with the sham‐treated controls. These results suggest that 30 mW/cm² (SAR 14.1 W/kg) microwave radiation can result in the perturbation of the synaptic vesicles associated proteins: synapsin I, synaptophysin, VAMP‐2, and syntaxin. The perturbation could induce the deposit of synaptic vesicle, which might be relative to the dysfunction of the synaptic transmission, even the cognition deficit. Synapse 63:1010–1016, 2009. © 2009 Wiley‐Liss, Inc.     

Exercise differentially regulates synaptic proteins associated to the function of BDNF

We explored the capacity of exercise to impact select events comprising synaptic transmission under the direction of brain-derived neurotrophic factor (BDNF), which may be central to the events by which exercise potentiates synaptic function. We used a specific immunoadhesin chimera (TrkB-IgG) that mimics the BDNF receptor, TrkB, to selectively block BDNF in the hippocampus during 3 days of voluntary wheel running. We measured resultant synapsin I, synaptophysin, and syntaxin levels involved in vesicular pool formation, endocytosis, and exocytosis, respectively. Synapsin I is involved in vesicle pool formation and neurotransmitter release, synaptophysin, in the biogenesis of synaptic vesicles and budding, and syntaxin, in vesicle docking and fusion. Exercise preferentially increased synapsin I and synaptophysin levels, without affecting syntaxin. There was a positive correlation between synapsin I and synaptophysin in exercising rats and synapsin I with the amount of exercise. Blocking BDNF abrogated the exercise-induced increases in synapsin I and synatophysin, revealing that exercise regulates select properties of synaptic transmission under the direction of BDNF.     

Distribution of Rab3a in rat nervous system: comparison with other synaptic vesicle proteins and neuropeptides

In the present study we have investigated the distribution of Rab3a in rat peripheral nervous system and compared it with the distribution of other synaptic vesicle proteins (synaptophysin, synapsin I), neuropeptides (CGRP, SP, NPY) and tyrosine hydroxylase (TH). Rab3a immunoreactivity (-IR) was always colocalized with synaptophysin-IR and synapsin I-IR in nerve terminals of the spinal cord and peripheral nerve endings. In many cases, Rab3a-IR was also present in the same axons and terminals as peptides. In crushed sciatic nerve axons, Rab3a was colocalized, proximal to the crush, with synaptophysin-IR, synapsin MR, CGRP-IR, and TH-IR, but only partially co-localized with NPY-IR and SP-IR. In the area distal to the crush, Rab3a-IR was very weakly positive in a few thin axons, while larger amount of synaptophysin, CGRP, NPY and SP immunoreactivities were detected. The subcellular distribution of peptides and Rab3a differed in that peptides were observed mainly in large granular structures, while Rab3a-IR was observed mainly as diffuse, finely granular immunoreactivity, in addition to a few exceptional large granules present in some axons. The results demonstrate that Rab3a is widely distributed in different types of neurons, i.e. motor, sensory, autonomic adrenergic and cholinergic neurons, and colocalized with other synaptic vesicle proteins, suggesting that Rab3a may play an essential role in neuronal function. Furthermore, Rab3a is present in many peptide containing axons and terminals, but with an apparently different subcellular distribution, being affiliated mostly with small synaptic vesicles and only occasionally with large vesicles, that may represent peptide contained vesicles.     

Increase in synaptic vesicle proteins in synapsin-transfected NG108-15 cells: A subcellular fractionation study

We have examined the subcellular distribution of synapsins and synaptophysin in density gradients from synapsin- and vector-transfected NGlO8-15 cells, since we recently found that transfection of synapsin IIb cDNA into neuroblastoma X glioma hybrid cells (NG108-15) resulted in cell lines that had a more neuronal phenotype than controls. The increase in synapsins and synaptophysin in the transfected cells was maximal in the region of the gradient containing small synaptic vesicles. The transferrin receptor, a marker for early endosomes, did not increase in the synapsin-containing fractions in the transfected cells. Secretogranin I, a soluble protein stored in and secreted from large dense cored vesicles, showed a very pronounced increase in the dense regions of gradients from transfected cells. These subcellular fractionation data suggest a possible role for the synapsins in the regulation of synaptic vesicle function. © 1995 Wiley-Liss, Inc.     

Clathrin-coated vesicles from bovine brain contain uncoupled GABAA receptors

Clathrin-coated vesicles are thought to be a vehicle for the sequestration of GABA_A receptors. For coated vesicles from bovine cerebrum, we examined the binding properties of [³H]muscimol, a GABA_A-specific agonist, [³H]flunitrazepam, a benzodiazepine agonist, and [³⁵S]t-butylbiocyclophosphorthionate (TBPS), a ligand for GABA_A receptor channels. Under standard conditions, the binding level of [³H]muscimol, [³H]flunitrazepam, and [³⁵S]TBPS to coated vesicles represented 12.3±1.8%, 7.9±1%, and 10.2±1.8%, respectively, of that in crude synaptic membranes. Coated vesicles showed a single [³H]flunitrazepam binding site with a K_D value (12 nM) which was 9-fold that for synaptic membranes. The allosteric coupling between binding sites was measured by the addition of GABA to [³H]flunitrazepam and [³⁵S]TBPS binding assays. For [³H]flunitrazepam binding to synaptic membranes, GABA gave an EC₅₀=2.0 μM and at saturation (100 μM) an enhancement of 122%. This stimulation was completely blocked by the GABA antagonist SR95531. In contrast, neither GABA nor SR95531 had a significant effect on [³H]flunitrazepam binding to CCVs, indicating that the allosteric interaction between GABA and benzodiazepine binding sites is abolished. Likewise, GABA displaced nearly all of the [³⁵S]TBPS binding to synaptic membranes but had no effect on binding to coated vesicles, indicating that coupling between the GABA binding sites and chloride channel is also impaired. Thus GABA_A receptors appear to be uncoupled during normal intracellular trafficking via coated vesicles. The presence of major GABA_A receptor subunits on these particles was verified by quantitative immunoblotting. Relative to the levels in synaptic membranes, CCVs contained 110±14% and 29.5±3.8%, respectively, of the immunoreactivity for GABA_A receptor β2 and α1 subunits. Thus, in comparison to GABA_A receptors on synaptic membranes, those on CCVs have a reduced α1/β2-subunit ratio. It may be suggested that a selective decline in the content of α1 subunits in coated vesicles could in part account for GABA_A receptor uncoupling.     

Phosphorylation-dependent Effects of Synapsin IIa on Actin Polymerization and Network Formation

The synapsins are a family of synaptic vesicle phosphoproteins which play a key role in the regulation of neurotransmitter release and synapse formation. In the case of synapsin I, these biological properties have been attributed to its ability to interact with both synaptic vesicles and the actin-based cytoskeleton. Although synapsin II shares some of the biological properties of synapsin I, much less is known of its molecular properties. We have investigated the interactions of recombinant rat synapsin Ila with monomeric and filamentous actin and the sensitivity of those interactions to phosphorylation, and found that: i) dephosphotylated synapsin II stimulates actin polymerization by binding to actin monomers and forming actively elongating nuclei and by facilitating the spontaneous nucleation/elongation processes; ii) dephosphorylated synapsin II induces the formation of thick and ordered bundles of actin filaments with greater potency than synapsin I; iii) phosphorylation by protein kinase A markedly inhibits the ability of synapsin II to interact with both actin monomers and filaments. The results indicate that the interactions of synapsin II with actin are similar but not identical to those of synapsin I and suggest that synapsin II may play a major structural role in mature and developing nerve terminals, which is only partially overlapping with the role played by synapsin I.     

Deletion of synapsins I and II genes alters the size of vesicular pools and rabphilin phosphorylation

Previous studies established that genetic deletion of synapsins, synaptic vesicle-associated phosphoproteins that regulate neurotransmitter release, decreases the number of synaptic vesicles in nerve terminals. To investigate whether these changes affect the release properties of the remaining synaptic vesicles, we used a radioactive labeling technique to measure release independently of the total number of synaptic vesicles. ³H-glutamate and ¹⁴C-γ-amino-butyric-acid (GABA) release from isolated nerve terminals prepared from the neocortex of synapsins I and II double knock-out mice (DKO) was assayed and compared to wild-type preparations. Hyperosmotic shock-evoked ³H-glutamate was reduced by 20 ± 3% from DKO nerve terminals and potassium depolarization-evoked glutamate release was also decreased by 28 ± 2%. Surprisingly, sucrose or potassium depolarization-evoked release of ¹⁴C-GABA was increased by 32 ± 4% and 29 ± 5%, respectively. The basal efflux of both ³H-glutamate and ¹⁴C-GABA increased by 17 ± 2% and 12 ± 2% from DKO nerve terminals. As lack of synapsins I and II, major phosphoproteins of synaptic vesicles, may lead to deregulation of phosphorylation events, we compared phosphorylation state of another synaptic vesicle protein, rabphilin. In DKO nerve terminals, membrane-associated rabphilin level was reduced by ∼0.28-fold, its phosphorylation at ²³⁴serine was increased by ∼1.61-fold whereas cytosolic rabphilin levels showed both more dramatic reduction in abundance, ∼16.5-fold, and increase in phosphorylation, ∼4.8-fold. Collectively, these data suggest that deletion of major synapsin isoforms leads to (1) deregulation of basal neurotransmission causing “leaky” basal release, (2) changes in either the size or mobilization of releasable or reserve pools, and (3) a decrease in rabphilin abundance accompanied by an increase in basal phosphorylation of the remaining rabphilin.     

Endogenous ligands for the quisqualate receptor: presence in rat brain cortex synaptic vesicles

The presence in highly purified rat brain cortex synaptic vesicles of endogenous ligands for rat brain quisqualate receptors was investigated. The vesicles were extracted, and their contents fractionated by high voltage electrophoresis. Endogenous ligands were detected by a radioreceptor assay in which such ligands competed with 50 nM -[³H]glutamate for binding to quisqualate receptors present in rat brain postsynaptic densities (PSDs). Binding of -[³H]glutamate to (NMDA) receptors, also present in PSDs, was blocked by 100 μM NMDA. We found that the endogenous ligands present in brain cortex synaptic vesicles for quisqualate receptors, were glutamate and aspartate, in a molar ratio of about two to one. The quisqualate receptor had an affinity 130-fold higher for glutamate (K_d 0.3 μM) than for aspartate, and the latter amino acid also showed a marked negative cooperativity for binding (Hill number 0.29, against 0.67 for glutamate). These findings suggest that glutamate is the natural transmitter that activates quisqualate receptors at some central excitatory synapses, and also that aspartate may be a classical transmitter, the receptor for which still remains to be shown.     

Translational suppression of calpain I reduces NMDA-induced spectrin proteolysis and pathophysiology in cultured hippocampal slices

Transfection of cultured hippocampal slices for five days with antisense oligonucleotides directed against mRNA encoding calpain I resulted in an approximately 60% decrease in the amount of caseinolytic activity stimulated by 10 μM calcium. Increases in a single proteolytic fragment of spectrin produced by 10–20 min of NMDA receptor stimulation were substantially ( 50%) reduced in antisense treated slices; this effect was not obtained in slices exposed to NMDA for 45 min. Attenuation of NMDA receptor-induced spectrin proteolysis by the antisense oligonucleotides was confirmed in immunoassays using antibodies that recognize multiple spectrin breakdown products and in immunocytochemical experiments with an antibody that defects an individual calpain I-mediated fragment. Translational suppression of calpain I did not detectably affect evoked synaptic responses but markedly improved their recovery from a 15 min infusion of NMDA. These results indicate that spectrin breakdown products provide a useful index of in situ calpain I activity and support the hypothesis that the protease plays a significant role in excitoxicity.     

The synaptic CT carbohydrate modulates binding and expression of extracellular matrix proteins in skeletal muscle: Partial dependence on utrophin

The CT carbohydrate, Neu5Ac/Neu5Gcα2,3[GalNAcβ1,4]Galβ1,4GlcNAcβ-, is specifically expressed at the neuromuscular junction in skeletal myofibers of adult vertebrates. When Galgt2, the glycosyltransferase that creates the synaptic β1,4GalNAc portion of this glycan, is overexpressed in extrasynaptic regions of the myofiber membrane, α dystroglycan becomes glycosylated with the CT carbohydrate and this coincides with the ectopic expression of synaptic dystroglycan-binding proteins, including laminin α4, laminin α5, and utrophin. Here we show that both synaptic and extrasynaptic forms of laminin and agrin have increased binding to the CT carbohydrate compared to sialyl-N-acetyllactosamine, its extrasynaptically expressed precursor. Muscle laminins also show increased binding to CT-glycosylated muscle α dystroglycan relative to its non-CT-containing glycoforms. Overexpression of Galgt2 in transgenic mouse skeletal muscle increased the mRNA expression of extracellular matrix (ECM) genes, including agrin and laminin α5, as well as utrophin, integrin α7, and neuregulin. Increased expression of ECM proteins in Galgt2 transgenic skeletal muscles was partially dependent on utrophin, but utrophin was not required for Galgt2-induced changes in muscle growth or neuromuscular development. These experiments demonstrate that overexpression of a synaptic carbohydrate can increase both ECM binding to α dystroglycan and ECM expression in skeletal muscle, and they suggest a mechanism by which Galgt2 overexpression may inhibit muscular dystrophy and affect neuromuscular development.