Michael J. Werle

Distinct target-derived signals organize formation, maturation, and maintenance of motor nerve terminals

Target-derived factors organize synaptogenesis by promoting differentiation of nerve terminals at synaptic sites. Several candidate organizing molecules have been identified based on their bioactivities in vitro, but little is known about their roles in vivo. Here, we show that three sets of organizers act sequentially to pattern motor nerve terminals: FGFs, beta2 laminins, and collagen alpha(IV) chains. FGFs of the 7/10/22 subfamily and broadly distributed collagen IV chains (alpha1/2) promote clustering of synaptic vesicles as nerve terminals form. beta2 laminins concentrated at synaptic sites are dispensable for embryonic development of nerve terminals but are required for their postnatal maturation. Synapse-specific collagen IV chains (alpha3-6) accumulate only after synapses are mature and are required for synaptic maintenance. Thus, multiple target-derived signals permit discrete control of the formation, maturation, and maintenance of presynaptic specializations.

Regulation of the Rapsyn Promoter by Kaiso and δ-Catenin

ABSTRACT Rapsyn is a synapse-specific protein that is required for clustering acetylcholine receptors at the neuromuscular junction. Analysis of the rapsyn promoter revealed a consensus site for the transcription factor Kaiso within a region that is mutated in a subset of patients with congenital myasthenic syndrome. Kaiso is a POZ-zinc finger family transcription factor which recognizes the specific core consensus sequence CTGCNA (where N is any nucleotide). Previously, the only known binding partner for Kaiso was the cell adhesion cofactor, p120 catenin. Here we show that δ-catenin, a brain-specific member of the p120 catenin subfamily, forms a complex with Kaiso. Antibodies against Kaiso and δ-catenin recognize proteins in the nuclei of C2C12 myocytes and at the postsynaptic domain of the mouse neuromuscular junction. Endogenous Kaiso in C2C12 cells coprecipitates with the rapsyn promoter in vivo as shown by chromatin immunoprecipitation assay. Minimal promoter assays demonstrated that the rapsyn promoter can be activated by Kaiso and δ-catenin; this activation is apparently muscle specific. These results provide the first experimental evidence that rapsyn is a direct sequence-specific target of Kaiso and δ-catenin. We propose a new model of synapse-specific transcription that involves the interaction of Kaiso, δ-catenin, and myogenic transcription factors at the neuromuscular junction.

Matrix metalloproteinase-3 removes agrin from synaptic basal lamina

Agrin, a heparin sulfate proteoglycan, is an integral member of the synaptic basal lamina and plays a critical role in the formation and maintenance of the neuromuscular junction. The N-terminal region of agrin binds tightly to basal lamina, while the C-terminal region interacts with a muscle-specific tyrosine kinase (MuSK) to induce the formation of the postsynaptic apparatus. Although the binding of agrin to basal lamina is tight, the binding of agrin to MuSK has yet to be shown; therefore, basal lamina binding is critical for maintaining the presentation of agrin to MuSK. Here we report evidence that supports our hypothesis that matrix metalloproteinase-3 (MMP-3) is responsible for the removal of agrin from synaptic basal lamina. Antibodies to the hinge region of human MMP-3 recognize molecules concentrated at the frog neuromuscular junction in both cross sections and whole mounts. Electron microscopy of neuromuscular junctions stained with antibodies to MMP-3 reveals that staining is found in the extracellular matrix surrounding the Schwann cell. Treatment of sections from frog anterior tibialis muscle with MMP-3 results in a clear and reproducible removal of agrin immunoreactivity from synaptic basal lamina. The same MMP-3 treatment does not alter anti-laminin staining. These results support our hypothesis that synaptic activity results in the activation of MMP-3 at the neuromuscular junction and that MMP-3 specifically removes agrin from synaptic basal lamina.

The presence of the casein kinase II phosphorylation sites of Vpu enhances the CD4+ T cell loss caused by the simian–human immunodeficiency virus SHIVKU-lbMC33 in pig-tailed macaques

The simian-human immunodeficiency virus (SHIV)/ macaque model for human immunodeficiency virus type 1 has become a useful tool to assess the role of Vpu in lentivirus pathogenesis. In this report, we have mutated the two phosphorylated serine residues of the HIV-1 Vpu to glycine residues and have reconstructed a SHIV expressing this nonphosphorylated Vpu (SHIV(S52,56G)). Expression studies revealed that this protein was localized to the same intracellular compartment as wild-type Vpu. To determine if this virus was pathogenic, four pig-tailed macaques were inoculated with SHIV(S52,56G) and virus burdens and circulating CD4(+) T cells monitored up to 1 year. Our results indicate that SHIV(S52,56G) caused rapid loss in the circulating CD4(+) T cells within 3 weeks of inoculation in one macaque (CC8X), while the other three macaques developed no or gradual numbers of CD4(+) T cells and a wasting syndrome. Histological examination of tissues revealed that macaque CC8X had lesions in lymphoid tissues (spleen, lymph nodes, and thymus) that were typical for macaques inoculated with pathogenic parental SHIV(KU-1bMC33) and had no lesions within the CNS. To rule out that macaque CC8X had selected for a virus in which there was reversion of the glycine residues at positions 52 and 56 to serine residues and/or compensating mutations occurred in other genes associated with CD4 down-regulation, sequence analysis was performed on amplified vpu sequences isolated from PBMC and from several lymphoid tissues at necropsy. Sequence analysis revealed a reversion of the glycine residues back to serine residues in this macaque. The other macaques maintained low virus burdens, with one macaque (P003) developing a wasting syndrome between months 9 and 11. Histological examination of tissues from this macaque revealed a thymus with severe atrophy that was similar to that of a previously reported macaque inoculated with a SHIV lacking vpu (Virology 293, 2002, 252). Sequence analysis revealed no reversion of the glycine residues in the vpu sequences isolated from this macaque. These results contrast with those from four macaques inoculated with the parental pathogenic SHIV(KU-1bMC33), all of which developed severe CD4(+) T cell loss within 1 month after inoculation. Taken together, these results indicate that casein kinase II phosphorylation sites of Vpu contributes to the pathogenicity of the SHIV(KU-1bMC33) and suggest that the SHIV(KU-1bMC33)/pig-tailed macaque model will be useful in analyzing amino acids/domains of Vpu that contribute to the pathogenesis of HIV-1.

Nitric oxide is a downstream mediator of agrin-induced acetylcholine receptor aggregation.

The synaptic basal lamina protein, agrin, is required for the formation of the neuromuscular junction. Agrin signals through a muscle-specific receptor tyrosine kinase (MuSK) initiating a cascade of events that lead to the aggregation of acetylcholine receptors (AChR) at the postsynaptic site. Another important synaptic signalling molecule is nitric oxide (NO), which is produced by the enzyme, nitric oxide synthase (NOS). We investigated the interaction between the agrin signalling cascade and the NO signalling cascade by treating cultured myotubes with agrin, NOS inhibitors, and NO donors. NOS inhibitors prevented agrin induced AChR aggregation and phosphorylation of the AChR beta subunit. Furthermore, NO donors induced AChR aggregation in the absence of agrin, as well as phosphorylation of the AChR beta subunit. These results demonstrate a role for NO as a downstream mediator of agrin induced AChR aggregation and AChR beta subunit phosphorylation at the neuromuscular junction.

Expression of frequenin at the frog (Rana) neuromuscular junction, muscle spindle and nerve.

Frequenin is a calcium binding protein previously implicated in the regulation of neurotransmission in Drosophila and Xenopus. We have used the frog (Rana pipiens) to study the localization and regulation of expression of frequenin-like molecules in the vertebrate peripheral nervous system. Affinity purified antibodies to frequenin recognize molecules in the neuromuscular junction, axons in the peripheral nerve, and neuronal processes in muscle spindles. Western blots of endplate regions, peripheral nerve, and brain, resulted in the labelling of a single 24 kDa band, which is the expected size for frequenin. These results suggest that frequenin expression is high in the frog peripheral nervous system, and may reflect a function for frequenin in synaptic transmission in vertebrates.

Autophagic Clearance of Sin Nombre Hantavirus Glycoprotein Gn Promotes Virus Replication in Cells

ABSTRACT Hantavirus glycoprotein precursor (GPC) is posttranslationally cleaved into two glycoproteins, Gn and Gc. Cells transfected with plasmids expressing either GPC or both Gn and Gc revealed that Gn is posttranslationally degraded. Treatment of cells with the autophagy inhibitors 3-methyladenine, LY-294002, or Wortmanin rescued Gn degradation, suggesting that Gn is degraded by the host autophagy machinery. Confocal microscopic imaging showed that Gn is targeted to autophagosomes for degradation by an unknown mechanism. Examination of autophagy markers LC3-I and LC3-II demonstrated that both Gn expression and Sin Nombre hantavirus (SNV) infection induce autophagy in cells. To delineate whether induction of autophagy and clearance of Gn play a role in the virus replication cycle, we downregulated autophagy genes BCLN-1 and ATG7 using small interfering RNA (siRNA) and monitored virus replication over time. These studies revealed that inhibition of host autophagy machinery inhibits Sin Nombre virus replication in cells, suggesting that autophagic clearance of Gn is required for efficient virus replication. Our studies provide mechanistic insights into viral pathogenesis and reveal that SNV exploits the host autophagy machinery to decrease the intrinsic steady-state levels of an important viral component for efficient replication in host cells.

ANTI-AGRIN STAINING IS ABSENT AT ABANDONED SYNAPTIC SITES OF FROG NEUROMUSCULAR JUNCTIONS

The neuromuscular junction is a plastic structure and is constantly undergoing changes as the nerve terminals that innervate the muscle fiber extend and retract their processes. In vivo observations on developing mouse neuromuscular junctions revealed that prior to the retraction of a nerve terminal the acetylcholine receptors (AChRs) under that nerve terminal disperse. Agrin is a protein released by nerve terminals that binds to synaptic basal lamina and directs the aggregation of AChRs and acetylcholinesterase (AChE) in and on the surface of the myotube. Thus, if the AChRs under a nerve terminal disperse, then the cellular signaling mechanism by which agrin maintains the aggregation of those AChRs, must have been disrupted. Two possibilities that could lead to the disruption of the agrin induced aggregation are that agrin is present at the synaptic basal lamina but is unable to direct the aggregation of AChRs, or that agrin has been removed from the synaptic basal lamina. Thus, if agrin were blocked, one would expect to see anti-agrin staining at abandoned synaptic sites; whereas if agrin were removed, anti-agrin staining would be absent at abandoned synaptic sites. We find that anti-agrin staining and alpha-bungarotoxin staining are absent at abandoned synaptic sites. Further, in vivo observations of retracting nerve terminals confirm that agrin is removed from the synaptic basal lamina within 7 days. Thus, while agrin will remain bound to synaptic basal lamina for months following denervation, it is removed within days following synaptic retraction.

Cell‐to‐Cell Signaling at the Neuromuscular Junction

The extracellular matrix at the neuromuscular junction plays many roles. The matrix plays a structural role in that it maintains the spatial relationship between the muscle cell, Schwann cell, and presynaptic motor neuron. The matrix also plays a role in cell‐to‐cell signaling. The most studied member of this group is the heparan sulfate proteoglycan, agrin. Agrin is an integral member of the synaptic matrix, and it plays the pivotal role of instructing the muscle cell to aggregate acetylcholine receptors (AChRs) to the synapse. Agrin is released by the motor neuron, where it binds stably to the extracellular matrix. Agrin interacts with the muscle‐specific tyrosine kinase (MuSK). Mice that lack agrin, or MuSK, fail to form neuromuscular junctions. Thus, the extracellular matrix is critical to both the structure and function of the neuromuscular junction.

Agrin-induced AChR aggregate formation requires cGMP and aggregate maturation requires activation of cGMP-dependent protein kinase

Previously, it was demonstrated that agrin acting through the gaseous, signaling molecule, nitric oxide (NO), induces the formation of AChR aggregates on myotubes in culture. Soluble guanylyl cyclase (sGC), which is present at the neuromuscular junction, is a common target of NO. Therefore, we hypothesized that sGC and cGMP are involved in the agrin signaling cascade. Inhibition of sGC hindered AChR aggregation in both agrin- and NO donor-treated cultured myotubes; whereas, a cGMP analogue was able to induce the formation of AChR aggregates on naïve muscle cells. Due to the presence of cyclic GMP-dependent protein kinase (PKG) at the neuromuscular junction, we tested the ability of a PKG inhibitor to alter the agrin signaling cascade. PKG inhibition did not prevent nascent AChR aggregate formation; however, these aggregates were diffuse and composed of numerous microaggregates consistent with incomplete maturation. Thus, we conclude that cGMP is important for the initiation of AChR aggregation, while PKG is involved in the maturation of AChR aggregates.