Combination of agrin and laminin increase acetylcholine receptor clustering and enhance functional neuromuscular junction formation in vitro

Bill G X Zhang, Anita Quigley, Justin Bourke, Cameron Nowell, Damian E. Myers, Peter F M Choong, Robert M I Kapsa

Research output: Contribution to journalArticleResearchpeer-review

20 Citations (Scopus)

Abstract

Clustering of acetylcholine receptors (AChR) at the postsynaptic membrane is a crucial step in the development of neuromuscular junctions (NMJ). During development and after denervation, aneural AChR clusters form on the sarcolemma. Recent studies suggest that these receptors are critical for guiding and initiating synaptogenesis. The aim of this study is to investigate the effect of agrin and laminin-1; agents with known AChR clustering activity; on NMJ formation and muscle maturation. Primary myoblasts were differentiated in vitro on collagen, laminin or collagen and laminin-coated surfaces in the presence or absence of agrin and laminin. The pretreated cells were then subject to innervation by PC12 cells. The number of neuromuscular junctions was assessed by immunocytochemical co-localization of AChR clusters and the presynaptic marker synaptophysin. Functional neuromuscular junctions were quantitated by analysis of the level of spontaneous as well as neuromuscular blocker responsive contractile activity and muscle maturation was assessed by the degree of myotube striation. Agrin alone did not prime muscle for innervation while a combination of agrin and laminin pretreatment increased the number of neuromuscular junctions formed and enhanced acetylcholine based neurotransmission and myotube striation. This study has direct clinical relevance for treatment of denervation injuries and creating functional neuromuscular constructs for muscle tissue repair. 

Original languageEnglish
Pages (from-to)551-565
Number of pages15
JournalDevelopmental Neurobiology
Volume76
Issue number5
DOIs
Publication statusPublished - 1 May 2016
Externally publishedYes

Keywords

  • acetylcholine receptors
  • agrin
  • laminin
  • neuromuscular junction
  • prepatterning

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