Biomedical subjects

M J Werle

Publications and source records attributed to M J Werle.

15 recordsLinked to original sources

PubMed2003

Structural alterations at the neuromuscular junctions of matrix metalloproteinase 3 null mutant mice.

Matrix metalloproteinases are important regulators of extracellular matrix molecules and cell-cell signaling. Antibodies to matrix metalloproteinase 3 (MMP3) recognize molecules at the frog neuromuscular junction, and MMP3 can remove agrin from synaptic basal lamina (VanSaun & Werle, 2000). To gain insight into the possible roles of MMP3 at the neuromuscular junction, detailed observations were made on the structure and function of the neuromuscular junctions in MMP3 null mutant mice. Striking differences were found in the appearance of the postsynaptic apparatus of MMP3 null mutant mice. Endplates had an increased volume of AChR stained regions within the endplate structure, leaving only small regions devoid of AChRs. Individual postsynaptic gutters were wider, containing prominent lines that represent the AChRs concentrated at the tops of the junctional folds. Electron microscopy revealed a dramatic increase in the number and size of the junctional folds, in addition to ectopically located junctional folds. Electrophysiological recordings revealed no change in quantal content or MEPP frequency, but there was an increase in MEPP rise time in a subset of endplates. No differences were observed in the rate or extent of developmental synapse elimination. In vitro cleavage experiments revealed that MMP3 directly cleaves agrin. Increased agrin immunofluorescence was observed at the neuromuscular junctions of MMP3 null mutant mice. These results provide strong evidence that MMP3 is involved in the control of synaptic structure at the neuromuscular junction and they support the hypothesis that MMP3 is involved in the regulation of agrin at the neuromuscular junction.

Agrin↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Agrin↗

PubMed2000

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.

Agrin↗

PubMed1999

Aggregates of acetylcholine receptors are not observed under anti-agrin staining schwann cell processes at the frog neuromuscular junction.

The frog neuromuscular junction offers a unique structure in which to observe fine details in the relationship between the motor neuron, muscle, and Schwann cell, which together comprise the neuromuscular junction. Schwann cell processes that extended from the synapse stained positively with anti-agrin antibodies. Immunocytochemistry revealed strong anti-agrin staining of the extracellular matrix surrounding the entire Schwann cell and the Schwann cell processes come in close contact with the muscle fiber. Dual-labeling experiments revealed a lack of acetylcholine receptor (AChR) aggregates on the surface of the muscle fiber directly under these anti-agrin-staining Schwann cell processes. The cDNA that codes for the C-terminal portion of agrin in frog (Rana pipiens) was cloned and sequenced. Polymerase chain reaction of frog brain, nerve, and muscle cDNA revealed that like other species, agrin transcripts that contain the B8, B11, or B19 inserts were observed only in brain tissue. Unlike other species, the exact site of the B inserts in frog was slightly altered, with the B insertion site occurring within a codon sequence. Our data are consistent with the hypothesis that Schwann cells produce agrin which lacks inserts at the B region, and that agrin lacking B inserts does not direct the aggregation of AChRs in vivo under physiological conditions and concentrations.

Agrin↗

PubMed1998

Agrin and acetylcholine receptor distribution following electrical stimulation.

Electrical stimulation is a therapeutic modality available for the preservation of muscle function following peripheral nerve injury. Agrin, a synaptic basal lamina protein, induces accumulation of acetylcholine receptors (AChRs) and other molecules at the neuromuscular junction. Electrical stimulation of denervated muscle does not alter agrin and AChR distribution at abandoned synaptic sites, supporting the hypothesis that the existing aggregation of synaptic molecules, which may be necessary for successful reinnervation, is unaltered by electrical stimulation of denervated muscle.

Animals↗

PubMed1997

Agrin and acetylcholine receptors are removed from abandoned synaptic sites at reinnervated frog neuromuscular junctions.

Changes in the distribution of agrin and acetylcholine receptors (AChRs) were examined during reinnervation and following permanent denervation as a means of understanding mechanisms controlling the distribution of these molecules. Following nerve damage in the peripheral nervous system, regenerating nerve terminals preferentially return to previous synaptic sites leading to the restoration of synaptic activity. However, not all portions of original synaptic sites are reoccupied: Some of the synaptic sites are abandoned by both the nerve terminal and the Schwann cell. Abandoned synaptic sites contain agrin, AChRs, and acetylcholinesterase (AChE) without an overlying nerve terminal or Schwann cell providing a unique location to observe changes in the distribution of these synapse-specific molecules. The distribution of anti-agrin and AChR staining at abandoned synaptic sites was altered during the process of reinnervation, changing from a dense, wide distribution to a punctate, pale pattern, and finally becoming entirely absent. Agrin and AChRs were removed from abandoned synaptic sites in reinnervated frog neuromuscular junctions, while in contralateral muscles which were permanently denervated, anti-agrin and AChR staining remained at abandoned synaptic sites. Decreasing synaptic activity during reinnervation delayed the removal of agrin and AChRs from abandoned synaptic sites. Altogether, these results support the hypothesis that synaptic activity controls a cellular mechanism that directs the removal of agrin from synaptic basal lamina and the loss of agrin leads to the dispersal of AChRs.

Agrin↗

PubMed1996

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.

Acetylcholinesterase↗

PubMed1992

Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions.

To test the hypothesis that agrin mediates motor neuron-induced aggregation of acetylcholine receptors (AChRs) in skeletal muscle fibers and to determine whether the agrin active in this process is released by motor neurons, we raised polyclonal antibodies to purified ray agrin that blocked its receptor aggregating activity. When the antibodies were applied to chick motor neuron--chick myotube cocultures, they inhibited the formation of AChR aggregates at and near neuromuscular contacts, demonstrating that agrin plays a role in the induction of the aggregates. Rat motor neurons, like chick motor neurons, induce AChR aggregates on chick myotubes. This effect was not inhibited by our antibodies, indicating that, although the antibodies inhibited the activity of chick agrin, they did not have a similar effect on rat agrin. We conclude that agrin released by rat motor neurons induced the chick myotubes to aggregate AChRs.

Agrin↗

PubMed1992

Agrin isoforms and their role in synaptogenesis.

Agrin is thought to mediate the motor neuron-induced aggregation of synaptic proteins on the surface of muscle fibers at neuromuscular junctions. Recent experiments provide direct evidence in support of this hypothesis, reveal the nature of agrin immunoreactivity at sites other than neuromuscular junctions, and have resulted in findings that are consistent with the possibility that agrin plays a role in synaptogenesis throughout the nervous system.

Agrin↗

PubMed1991

Postmetamorphic development of neuromuscular junctions and muscle fibers in the frog cutaneous pectoris.

Synaptic size, synaptic remodelling, polyneuronal innervation, and synaptic efficacy of neuromuscular junctions were studied as a function of growth in cutaneous pectoris muscles of postmetamorphic Rana pipiens. Recently metamorphosed frogs grew rapidly, and this growth was accompanied by hypertrophy of muscle fibers, myogenesis, and increases in the size and complexity of neuromuscular junctions. There were pronounced gradients in pre- and postsynaptic size across the width of the muscle, with neuromuscular junctions and muscle fibers near the medial edge being smaller than in more lateral regions. The incidence of polyneuronal innervation, measured physiologically and histologically, was also higher near the medial edge. Growth-associated declines in all measures of polyneuronal innervation indicated that synapse elimination occurs throughout life. Electrophysiology also demonstrated regional differences in synaptic efficacy and showed that doubly innervated junctions have lower synaptic efficacy than singly innervated junctions. Repeated, in vivo observations revealed extensive growth and remodelling of motor nerve terminals and confirmed that synapse elimination is a slow process. It was concluded that some processes normally associated with embryonic development persist long into adulthood in frog muscles.

Animals↗

PubMed1991

Elevated levels of polyneuronal innervation persist for as long as two years in reinnervated frog neuromuscular junctions.

When the nerve to an adult frog sartorius muscle is crushed, and axons are allowed to regenerate, the level of polyneuronal innervation at reinnervated neuromuscular junctions is higher than normal. With time, much of this polyneuronal innervation is reduced by the process of synapse elimination (Werle and Herrera, 1988). Using intracellular recording, we estimated the level of polyneuronal innervation in adult frog (Rana pipiens) sartorius muscles 2 years (range: 1.7-2.4 years) after crushing the sartorius nerve. We found that 27% (S.E. = 1.4%) of the junctions in muscles 2 years after reinnervation were polyneuronally innervated, whereas only 10% (S.E. = 1.2%) of the junctions in normal frog muscles were polyneuronally innervated. Thus, the synapse elimination that occurs following reinnervation does not restore the normal level of polyneuronal innervation. Histological comparisons of junctional structure between muscles 2 years after reinnervation and normal muscles revealed substantial differences. Reinnervated junctions had a greater length of synaptic gutter apposed by nerve terminal processes, more axonal inputs, more empty synaptic gutter, more instances of single synaptic gutters innervated by more than one axon, and longer lengths of nerve terminal processes that connect synaptic gutters within a junction. On the basis of this physiological and anatomical evidence, we conclude that nerve injury causes persistent changes in the pattern of muscle innervation.

Animals↗

PubMed1990

Mechanisms of elimination, remodeling, and competition at frog neuromuscular junctions.

Mechanisms governing synapse elimination, synaptic remodeling, and polyneuronal innervation were examined in anatomical and electrophysiological studies of frog neuromuscular junctions. There was a substantial level of polyneuronal innervation in adult junctions and this varied seasonally. Nerve terminal retraction and synapse elimination occurred during normal growth and following reinnervation. Synapse elimination was not inevitable, however. Repeated in vivo observations of some identified junctions showed that polyneuronal innervation could persist for over a year, while at other junctions it arose de novo by terminal sprouting. We concluded that polyneuronal innervation in adult muscles was governed by an equilibrium between processes of retraction and elimination on one hand, and sprouting and synaptogenesis on the other. Other observations revealed that structural remodeling was a common feature of adult junctions. Most often, remodeling involved the simultaneous growth and retraction of different parts of the same junction. The net result was usually junctional growth that, in small frogs, appeared to provide a good match between synaptic size and the electrical demands of transmission. In larger animals, pre- and postsynaptic sizes were not as well matched, providing morphological evidence for a growth-associated decline in synaptic efficacy. Finally, electrophysiology was used to describe some of the functional correlates and consequences of competitive interactions between the terminals of different axons. These results are explained by a hypothetical mechanism that involves trophic support provided by the muscle to the motoneuron, the overall level of nerve-muscle activity, and the synchrony of pre- and postsynaptic activity.

Animals↗

PubMed1988

Synaptic competition and the elimination of polyneuronal innervation following reinnervation of adult frog sartorius muscles.

The elimination of polyneuronal innervation (synapse elimination) that occurs following reinnervation was studied in sartorius muscles of adult Rana pipiens. The percentage of neuromuscular junctions that were polyneuronally innervated declined from 47% at 40-80 days after nerve crush to 22% at greater than 250 days after nerve crush. We measured the size, synaptic strength, and position of competing nerve terminals at identified dually innervated neuromuscular junctions at these two different periods of synapse elimination. Our goal was to determine if any of these parameters play a role in the competition between nerve terminals that ultimately results in the elimination of polyneuronal innervation. Our data support the hypothesis that polyneuronal innervation will persist if competing nerve terminals are of similar synaptic efficacies but will be eliminated if the competing terminals are of different synaptic efficacies. We also tested, but failed to find any evidence, that the spatial proximity of competing nerve terminals at the same synaptic site influences the elimination of polyneuronal innervation.

Action Potentials↗

PubMed1987

Synaptic competition and the persistence of polyneuronal innervation at frog neuromuscular junctions.

Mechanisms governing the elimination of polyneuronal innervation were examined by correlating the morphology and physiology of competing nerve terminals at identified dually innervated neuromuscular junctions in sartorius muscles of adult frogs (Rana pipiens). Synaptic efficacy (endplate potential amplitude per unit nerve terminal length) was presumed to reflect the ability of a terminal to compete for synaptic space. The synaptic efficacies of two terminals at the same synaptic site were found to be surprisingly equal, with a median difference of 33%. Much more variation would be expected if dually innervated junctions were randomly innervated by pairs of terminals having the same range of synaptic efficacy as that found at singly innervated junctions in the same muscle. This finding supports the hypothesis that the weaker input is eliminated from dually innervated junctions when there is a large discrepancy in competitive efficacy, and that both inputs may persist if competitive efficacies are relatively equal. We also tested but failed to find support for the hypothesis that spatial proximity between competing terminals intensifies competition for synaptic space during synapse elimination.

Animals↗