Null alleles of agrin cause embryonic lethality with failed formation of neuromuscular junctions, but this F1061S point substitution in the SEA domain in the SN form of Agrin provides a viable model for congenital myasthenic syndrome type 8 (CMS8). Although no change was detected in transcript quantity or size and the alternative splicing at the Z splice site of exons 32 and 33 appears normal, this mutation is a partial loss of function hypomorph with decreased glycosylation of the mutant protein. Cell transfection studies show that this point mutation can decrease proteolysis of this protein by neurotrypsin and result in decreased externalization and secretion.
Agrnnmf380 homozygotes are generally smaller than littermate controls by 15 days of age, there is muscle stiffness and atrophy, with their hind limbs appearing to be too weak to carry the weight, remaining low during movement, and they generally die between a few weeks to a few months of age. Initial pathology assessment on three mutants (27 - 34 days of age) revealed neurogenic myopathy with small and regenerating muscle fibers. Toes, especially those of hind paws may not fully extend, and hind limbs may move together rather than alternate. Brief spasms of hind or front limbs can also be observed. When lifting the mice by their tail, hind limbs remain clasped to the body. They develop an intense body tremor during movement and at rest due to postnatal degradation of neuromuscular junctions that proceeds at different rates in different muscles. The onset of the outward phenotype is approximately 2 weeks of age, although some muscles show perinatal neuromuscular degeneration. Defects of the neuromuscular junctions include smaller, underdeveloped postsynaptic sites, diminished alpha-bungarotoxin staining indicating decreased acetylcholine esterase receptors, and motor nerve terminals that sprout beyond the endplates. Transmission electron microscopy of the neuromuscular junctions from the plantaris muscle at 20 days of age showed abnormalities including extensive subsynaptic reticulum not continuous with the plasma membrane and decreased acetylcholine esterase and beta-dystroglycan from the neuromuscular junction. Genetic background impacts the phenotype of Agrnnmf380, with the severity in homozygotes being similar when backcrossed to FVB/NJ, DBA/2J, or BALB/cJ, but much less severe when backcrossed to N3 on CAST/EiJ.
The degeneration of neuromuscular junctions in these mutants varies between different muscles. The diaphragm shows overgrowth of presynaptic endings and indistinct alpha-bungarotoxin staining by 1 day of age and almost no intact neuromuscular junctions by P14. The tibialis anterior appears normal in newborns, but by P14 disaggregating neuromuscular junctions are found. The neuromuscular junctions of the soleus appear normal at P4 but not by P14. The triangularis sterni show normal innervation at P9, but decreased alpha-bungarotoxin staining and abnormal presynaptic morphology by P13, and by P18 the neuromuscular junctions were found to be degraded both pre- and post-synaptically. However, the extraocular muscles do not show neuromuscular degeneration at P30. Assessment of the tibialis anterior and gastrocnemius muscles found that neuromuscular disaggregation and denervation occurs in the medial before the intermediate region. It is worthy of note that retraction or degeneration of the axons of peripheral nerves was not found. (Bogdanik and Burgess, 2011.)
