H. Benjamin Peng

Cortactin-Src Kinase Signaling Pathway Is Involved in N-syndecan-dependent Neurite Outgrowth

N-syndecan (syndecan-3) was previously isolated as a cell surface receptor for heparin-binding growth-associated molecule (HB-GAM) and suggested to mediate the neurite growth-promoting signal from cell matrix-bound HB-GAM to the cytoskeleton of neurites. However, it is unclear whether N-syndecan would possess independent signaling capacity in neurite growth or in related cell differentiation phenomena. In the present study, we have transfected N18 neuroblastoma cells with a rat N-syndecan cDNA and show that N-syndecan transfection clearly enhances HB-GAM-dependent neurite growth and that the transfected N-syndecan distributes to the growth cones and the filopodia of the neurites. The N-syndecan-dependent neurite outgrowth is inhibited by the tyrosine kinase inhibitors herbimycin A and PP1. Biochemical studies show that a kinase activity, together with its substrate(s), binds specifically to the cytosolic moiety of N-syndecan immobilized to an affinity column. Western blotting reveals both c-Src and Fyn in the active fractions. In addition, cortactin, tubulin, and a 30-kDa protein are identified in the kinase-active fractions that bind to the cytosolic moiety of N-syndecan. Ligation of N-syndecan in the transfected cells by HB-GAM increases phosphorylation of c-Src and cortactin. We suggest that N-syndecan binds a protein complex containing Src family tyrosine kinases and their substrates and that N-syndecan acts as a neurite outgrowth receptor via the Src kinase-cortactin pathway.

Cullin Mediates Degradation of RhoA through Evolutionarily Conserved BTB Adaptors to Control Actin Cytoskeleton Structure and Cell Movement

Cul3, a Cullin family scaffold protein, is thought to mediate the assembly of a large number of SCF (Skp1-Cullin1-F-box protein)-like ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors. Cul3 controls early embryonic development in several genetic models through mechanisms not understood. Very few functional substrate/adaptor pairs for Cul3 ubiquitin ligases have been identified. Here, we show that Cul3 knockdown in human cells results in abnormal actin stress fibers and distorted cell morphology, owing to impaired ubiquitination and degradation of small GTPase RhoA. We identify a family of RhoA-binding BTB domain adaptors conserved from insects to mammals, designated BACURDs. BACURDs form ubiquitin ligase complexes, which selectively ubiquitinate RhoA, with Cul3. Dysfunction of the Cul3/BACURD complex decreases cell migration potential and impairs RhoA-mediated convergent extension movements during Xenopus gastrulation. Our studies reveal a previously unknown mechanism for controlling RhoA degradation and regulating RhoA function in various biological contexts, which involves a Cul3/BACURD ubiquitin ligase complex.

The Relationship between Perlecan and Dystroglycan and its Implication in the Formation of the Neuromuscular Junction

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.

Differential Effects of Neurotrophins and Schwann Cell-Derived Signals on Neuronal Survival/Growth and Synaptogenesis

Recent studies have shown that the survival of mammalian motoneurons in vitro is promoted by neurotrophins (NTs) and cAMP. There is also evidence that neurotrophins enhance transmitter release. We thus investigated whether these agents also promote synaptogenesis. Cultured Xenopus spinal cord neurons were treated with a mixture of BDNF, glia-derived neurotrophic factor, NT-3, and NT-4, in addition to forskolin and IBMX or the cell-permeant form of cAMP, to elevate the cAMP level. The outgrowth and survival of neurons were dramatically increased by this trophic stimulation. However, when these neurons were cocultured with muscle cells, the trophic agents resulted in a failure of synaptogenesis. Specifically, the induction of ACh receptor (AChR) clustering in cultured muscle cells was inhibited at nerve—muscle contacts, in sharp contrast to control, untreated cocultures. Because AChR clustering induced by agrin or growth factor-coated beads in muscle cells was unaffected by trophic stimulation, its effect on synaptogenesis is presynaptic in origin. In the control, agrin was deposited along the neurite and at nerve—muscle contacts. This was significantly downregulated in cultures treated with trophic stimuli. Reverse transcriptase-PCR analyses showed that this decrease in agrin deposition was caused by an inhibition of agrin synthesis by trophic stimuli. Both agrin synthesis and induction of AChR clustering were restored under trophic stimulation when Schwann cell-conditioned medium was introduced. These results suggest that trophic stimulation maintains spinal neurons in the growth state, and Schwann cell-derived factors allow them to switch to the synaptogenic state.

Dynamics of synaptic vesicles in cultured spinal cord neurons in relationship to synaptogenesis

The dynamics of synaptic vesicles (SVs) during the development of presynaptic specializations in cultured Xenopus spinal cord neurons was studied with the fluorescent vesicular probe FM1-43. In naive neurons that have not contacted synaptic targets, packets of SVs are distributed along the entire neurite and are quite mobile. The interaction with the synaptic target, such as a muscle cell or a latex bead coated with basic fibroblast growth factor, results in the localization and immobilization of SV packets at the contact site. Depolarization resulted in exocytosis of SVs in both naive and target-contacted neurites. Okadaic acid, a phosphatase inhibitor, caused a dispersal of SV packets in both naive and target-contacted neurites. Thus, prior to target contact, SVs are already organized into packets capable of release and recycling by a phosphorylation-dependent mechanism. Target interaction then recruits and anchors these functional SV packets into forming the presynaptic nerve terminal. With fluorescent phalloidin as a probe, F-actin was found to colocalize with SV clusters at bead-neurite contacts. Although okadaic acid caused a dispersal of SVs at the beads, F-actin localization there was relatively resistant to this drug treatment. This suggests that SVs become localized at the target by interacting with an actin-based cytoskeletal specialization in a phosphorylation-sensitive manner. The induction of this cytoskeletal specialization by the target may be an early event in presynaptic differentiation.

A role of midkine in the development of the neuromuscular junction

Midkine (MK) is a member of a family of developmentally regulated neurotrophic and heparin-binding growth factors. It is expressed during the midgestation period in a retinoid-acid dependent manner during embryogenesis in the mouse. In vitro, it promotes neurite outgrowth from spinal cord neurons and cell migration. It expression is strongest in the central nervous system, thus suggesting a function for this protein in neural development. In this study, the role of MK in synaptogenesis was examined in the Xenopus system. A Xenopus MK cDNA was cloned from an embryonic library encompassing neurulation and synaptogenesis stages. By Northern blot analysis, MK mRNA was detected from the onset of neurulation and throughout the stages of synaptogenesis in the Xenopus embryo. This suggests that MK is also an important growth regulator in Xenopus embryogenesis. To study the function of MK in the development of the neuromuscular junction (NMJ), fusion proteins were made and their ability to induce the formation of acetylcholine receptor (AChR) clusters in cultured muscle cells was studied. Beads coated with MK strongly induce AChR clustering. When nerve-muscle cocultures were labeled with antibodies made against the MK fusion protein, MK immunoreactivity was detected at the NMJ. Unlike heparin-binding growth-associated molecule (HB-GAM), another member of this growth factor family, MK expression cannot be detected in the muscle but is present in spinal cord neurites. Consistent with these in vitro data is the observation that MK mRNA is only localized in the central nervous system but the protein is deposited at the intersomitic junction where the NMJ is located in vivo. Exogenously applied MK does bind to the heparan sulfate proteoglycan on the surface of Xenopus muscle cells. Agrin, a heparan-sulfate proteoglycan that induces the formation of AChR clusters in cultured muscle cells, binds strongly to MK. Bath application of MK in conjunction with agrin results in a change in the pattern of AChR clustering induced by agrin alone. These data suggest that MK is a neuron-derived factor that participates in the signal transduction process during NMJ development.

Regulation of a Transcript Encoding the Proline-rich Membrane Anchor of Globular Muscle Acetylcholinesterase THE SUPPRESSIVE ROLES OF MYOGENESIS AND INNERVATING NERVES

The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G4 AChE), was revealed in muscle during myogenic differentiation under the influence of innervation. During myotube formation of C2C12 cells, the expression of AChET protein and the enzymatic activity were dramatically increased, but the level of G4 AChE was relatively decreased. This G4 AChE in C2C12 cells was specifically recognized by anti-PRiMA antibody, suggesting the association of this enzyme with PRiMA. Reverse transcription-PCR analysis revealed that the level of PRiMA mRNA was reduced during the myogenic differentiation of C2C12 cells. Overexpression of PRiMA in C2C12 myotubes significantly increased the production of G4 AChE. The oligomerization of G4 AChE, however, did not require the intracellular cytoplasmic tail of PRiMA. After overexpressing the muscle regulatory factors, myogenin and MyoD, the expressions of PRiMA and G4 AChE in cultured myotubes were markedly reduced. In addition, calcitonin gene-related peptide, a known motor neuron-derived factor, and muscular activity were able to suppress PRiMA expression in muscle; the suppression was mediated by the phosphorylation of a cAMP-responsive element-binding protein. In accordance with the in vitro results, sciatic nerve denervation transiently increased the expression of PRiMA mRNA and decreased the phosphorylation of cAMP-responsive element-binding protein as well as its activator calcium/calmodulin-dependent protein kinase II in muscles. Our results suggest that the expression of PRiMA, as well as PRiMA-associated G4 AChE, in muscle is suppressed by muscle regulatory factors, muscular activity, and nerve-derived trophic factor(s).

The Role of an Agrin-Growth Factor Interaction in ACh Receptor Clustering

The clustering of acetylcholine receptors (AChRs) at the neuromuscular junction is mediated in part by the heparan-sulfate proteoglycan agrin. However, our previous studies have also suggested the role of heparin-binding growth-associated molecular (HB-GAM) in AChR clustering. Here the role of an agrin-HB-GAM interaction in this process was examined using cultured Xenopus muscle cells. Agrin-coated beads further treated with HB-GAM were highly effective in AChR cluster induction. Protein overlay assays showed specific binding of HB-GAM to agrin. In addition, agrin-enriched neuritic tracks bound HB-GAM in a manner that showed a high degree of colocalization between the neural agrin and the applied factor. Finally, the introduction of exogenous HB-GAM together with soluble agrin resulted in the appearance of AChR clusters on the dorsal surface of cells in an agrin isoform-dependent manner; a dramatic change from the characteristic ventral AChR clustering seen in response to agrin alone. These results suggest that agrin may mediate AChR clustering by interacting with muscle-bound heparin-binding growth factors such as HB-GAM.

Tyrosine phosphatase regulation of MuSK-dependent acetylcholine receptor clustering

During vertebrate neuromuscular junction (NMJ) development, nerve-secreted agrin induces acetylcholine receptor (AChR) clustering in muscle by activating the muscle-specific tyrosine kinase MuSK. Recently, it has been recognized that MuSK activation-dependent AChR clustering occurs in embryonic muscle even in the absence of agrin, but how this process is regulated is poorly understood. We report that inhibition of tyrosine phosphatases in cultured C2 mouse myotubes using pervanadate enhanced MuSK auto-activation and agrin-independent AChR clustering. Moreover, phosphatase inhibition also enlarged the AChR clusters induced by agrin in these cells. Conversely, in situ activation of MuSK in cultured Xenopus embryonic muscle cells, either focally by anti-MuSK antibody-coated beads or globally by agrin, stimulated downstream tyrosine phosphatases, which could be blocked by pervanadate treatment. Immunoscreening identified Shp2 as a major tyrosine phosphatase in C2 myotubes and down-regulation of its expression by RNA interference alleviated tyrosine phosphatase suppression of MuSK activation. Significantly, depletion of Shp2 increased both agrin-independent and agrin-dependent AChR clustering in myotubes. Our results suggest that muscle tyrosine phosphatases tightly regulate MuSK activation and signaling and support a novel role of Shp2 in MuSK-dependent AChR clustering.

Molecular regulation of postsynaptic differentiation at the neuromuscular junction

The neuromuscular junction (NMJ) is a synapse that develops between a motor neuron and a muscle fiber. A defining feature of NMJ development in vertebrates is the re‐distribution of muscle acetylcholine (ACh) receptors (AChRs) following innervation, which generates high‐density AChR clusters at the postsynaptic membrane and disperses aneural AChR clusters formed in muscle before innervation. This process in vivo requires MuSK, a muscle‐specific receptor tyrosine kinase that triggers AChR re‐distribution when activated; rapsyn, a muscle protein that binds and clusters AChRs; agrin, a nerve‐secreted heparan‐sulfate proteoglycan that activates MuSK; and ACh, a neurotransmitter that stimulates muscle and also disperses aneural AChR clusters. Moreover, in cultured muscle cells, several additional muscle‐ and nerve‐derived molecules induce, mediate or participate in AChR clustering and dispersal. In this review we discuss how regulation of AChR re‐distribution by multiple factors ensures aggregation of AChRs exclusively at NMJs. IUBMB Life, 57: 719‐730, 2005