Genetic Tests of the Agrin Hypothesis

The agrin hypothesis has also endured in vivo testing using genetic approaches in mice. Mice that lack agrin die at birth with nonfunctional NMJs10. During embryonic mouse development, motor neurons contact muscle fibers at approximately embryonic day 12-13 (E12-13). Examination of NMJs as early as E15 reveals that postsynaptic differentiation is nearly absent in mice lacking agrin.

This phenotype was initially described in mice in which the nervous-system-specific exons encoding the Z+ isoforms (the exons that make agrin active in AChR clustering assays) were deleted by homologous recombination. These mice also had severely reduced expression of all other agrin isoforms, including those forms expressed by muscle. The same phenotype was seen in other engineered alleles of agrin. Mice lacking all agrin after the fifth N-terminal follistatin repeat (Figure 1.2)11 and mice lacking only the Z-exons with normal expression of all other agrin isoforms12 have the same phenotype, a marked reduction in AChR clusters and postsynaptic differentiation, and an overgrowth of motor neurons well beyond their normal domain in the central end-plate band of the muscle.

A fourth allele of agrin confirmed the importance of agrin as a component of the ECM for NMJ formation13. Agrin has two alternative N-termini that result from transcripts with distinct transcriptional and translational start sites. One of these forms (LN agrin) is secreted and binds the y1 chains of laminin trimers. This is the form made by most non-neuronal cell types, and also by motor neurons. The second agrin isoform is a type-2 transmembrane protein that is the predominant form in most of the CNS14,15. The LN isoform of agrin was eliminated by a gene-trap insertion between the LN encoding exons and the SN exon. Therefore LN transcripts were selectively intercepted without disrupting the expression of the SN isoform. Mice homozygous for this gene-trap insertion lack agrin in basal laminae throughout the body and show an NMJ phenotype identical to that of other agrin mutants. However, SN agrin is still expressed in the CNS of these animals, and homogenates of CNS tissue are still active in AChR receptor clustering assays, while homogenates from mice lacking the bulk of the agrin coding sequence are not. These results indicate that transmembrane SN agrin is capable of inducing AChR clusters in vitro, but it is insufficient in vivo.

The function of SN agrin in the CNS is under investigation. Targeting the SN encoding exon results in mice that are viable despite an 80% reduction of agrin in the brain (our unpublished data). However, these mice have normal NMJ junction morphology and show no behavioral signs of neuromuscular dysfunction, indicating that transmembrane SN agrin is not necessary for NMJ formation, while the ECM-bound LN isoform is essential.

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