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Ann. N.Y. Acad. Sci. 956: 394–396 (2002). © 2002 New York Academy of Sciences.

Conservation of Synapse-Signaling Pathwaysat the Extraocular Muscle Neuromuscular Junction

SANGEETA KHANNAa AND JOHN D. PORTERa,b

Departments of aOphthalmology and bNeurology and Neurosciences, Case Western Reserve University, and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA

KEYWORDS: extraocular muscle; neuromuscular junction; synapse; signaltransduction

PURPOSE

The cell and molecular biology of extraocular muscle (EOM) is distinct from oth-er skeletal muscles.1,2 Novel aspects of the innervation pattern of EOM include: highmotoneuron discharge rates, presence of multiply innervated muscle fiber types inthe adult, retention of embryonic acetylcholine receptor isoforms at some neuromus-cular junctions (NMJs), absence of significant postjunctional membrane foldings atmost NMJs, and sensitivity of EOM to neuromuscular transmission disorders (my-asthenia gravis) and distinct response to botulinum toxin. The formation and matu-ration of the NMJ require a series of inductive interactions between axon and musclefiber. These interactions culminate in the juxtaposition of a highly specialized nerveterminal with an elaborate postsynaptic apparatus.3,4 The sarcolemmal DGC and itsassociated proteins are central in this process of synapse formation and stabiliza-tion.5,6 The central hypothesis of this work was that the unique phenotype, and func-tional properties, of EOM may require muscle group-specific adaptations toorganize and maintain the NMJ. Therefore, the signaling and sarcolemmal organiza-tion at the NMJ may differ between EOM singly (SIF) and multiply innervated fibertypes (MIF) and between EOM and other skeletal muscles.

METHODS

Here, we have examined cryostat sections of EOM of adult mice using immuno-histochemistry for the neuregulin/erbB and agrin/MuSK pathway molecules and im-portant components of the junctional DGC. The en-plaque (on SIFs) and the en-grappe junctions (on MIFs) were identified by their morphologic pattern using Tex-

Address for correspondence: Sangeeta Khanna, M.D., Research Institute, University Hospitalsof Cleveland, 11100 Euclid Avenue, Cleveland, Ohio 44106. Voice: 216-844-1429; fax: 216-844-4792.

sxk128@po.cwru.edu

395KHANNA & PORTER: EXTRAOCULAR MUSCLE NMJ

as-red conjugated bungarotoxin (Molecular Probes; 4 µg/ml), which labels theAChRs. The other proteins were detected using monoclonal and polyclonal antibod-ies specific for: utrophin (NCL-DRP2, Novocastra Labs), α1-syntrophin (258), β1-syntrophin (37) and β2-syntrophin (28), rapsyn (1234), α-dystrobrevins DB-1 (670)and DB-2 (DB2) (all from Stan Froehner, University of Washington), s-laminin (D7;Joshua Sanes, Washington University), neuregulin (NDF; Jeffrey A Loeb, WayneState University), erbB2 (Neu:sc-284; Santa Cruz Biotechnology, Inc. Santa Cruz),erbB3 and erbB4 (05-390, 06-572; Upstate Biotechnology, NY), agrin (AGR-550;StressGen Biotechnology Corp., Canada), and MuSK (Markus Ruegg, University ofBasel). Junctions were examined for the cellular localization and colocalization ofthese proteins with AChRs using a scanning laser confocal microscope.

RESULTS

The components of the junctional DGC and the agrin/MuSK and the neuregulin/erbB nerve-muscle signaling pathways that we examined, colocalized with NMJacetylcholine receptor aggregates in both the MIFs and the SIFs, similar to the typ-ical skeletal muscles. There were some differences in the extrasynaptic localization

TABLE 1. Comparison of the cellular localization of the synaptic molecules

Protein

EOM SIFs EOM MIFs Skeletal musclea

NMJExtra-

synaptic NMJExtra-

synaptic NMJExtra-

synaptic

agrin + − + − + −MuSK + − + − + −rapsyn + − + − + −neuregulin + − + − + −erbB2 + − + − + −erbB3 + − + − + −erbB4 + − + − + −utrophin + − + − + −α-syntrophin + + + + + +β1syntrophin + − + + + +

(fast fibers)7

β2syntrophin + − + − + −α-DB-1 + − + + + −6

α-DB-2 + + + + + +s-laminin + − + − + −

ABBREVIATIONS: EOM, extraocular muscle; MIFs, multiply innervated fiber types; NMJ, neu-romuscular junction; SIFs, singly innervated fiber types.

aSkeletal muscle data published elsewhere by several other groups.

396 ANNALS NEW YORK ACADEMY OF SCIENCES

of some DGC proteins, namely, β1-syntrophin and α-dystrobrevin1 on EOM MIFs.The results are summarized in TABLE 1.

CONCLUSIONS

The EOM neuromuscular junctions differ in morphologic pattern from that of thetypical NMJs, not only in the presence of MIFs in the adult but also in the SIF en-plaque junctions. However, we find here that these do not differ from the well-stud-ied pattern of signaling mechanisms of typical skeletal muscle NMJ. The key com-ponents of the neuregulin/erbB and agrin/MuSK pathways and the junctional DGCproteins are present at EOM NMJs, establishing that synapse formation/maintenancepathways are conserved in all mammalian skeletal muscles, including the phenotyp-ically novel EOMs. The inordinately high discharge rates of ocular motoneurons andthe differential involvement of EOM in several neuromuscular diseases, however,suggest that there are other, as yet unidentified, features of NMJs that are unique tothe extraocular muscle.

ACKNOWLEDGMENTS

We thank Denise Hatala for technical support. This work was supported by grantsfrom Knights-Templar Eye Research Foundation, Research to Prevent Blindness, theMuscular Dystrophy Association USA, the Evenor Armington Fund, and by NIHGrants EY09834, EY12779, and P30 EY11373.

REFERENCES

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