Modulation of the Acetylcholine Receptor Clustering Pathway Improves Neuromuscular Junction Structure and Muscle Strength in a Mouse Model of Congenital Myasthenic Syndrome.

Introduction: Congenital myasthenic syndromes (CMS) are a diverse group of inherited neuromuscular disorders characterized by a failure of synaptic transmission at the neuromuscular junction (NMJ). CMS often present early with fatigable weakness and can be fatal through respiratory complications. The AGRN gene is one of over 30 genes known to harbor mutations causative for CMS. In this study, we aimed to determine if a compound (NT1654), developed to stimulate the acetylcholine receptor (AChR) clustering pathway, would benefit a mouse model of CMS caused by a loss-of-function mutation in Agrn (Agrn nmf380 mouse). Methods: Agrn nmf380 mice received an injection of either NT1654 or vehicle compound daily, with wild-type litter mates used for comparison. Animals were weighed daily and underwent grip strength assessments. After 30 days of treatment animals were sacrificed, and muscles collected. Investigations into NMJ and muscle morphology were performed on collected tissue. Results: While minimal improvements in NMJ ultrastructure were observed with electron microscopy, gross NMJ structure analysis using fluorescent labelling and confocal microscopy revealed extensive postsynaptic improvements in Agrn nmf380 mice with NT1654 administration, with variables frequently returning to wild type levels. An improvement in muscle weight and myofiber characteristics helped increase forelimb grip strength and body weight. Conclusions: We conclude that NT1654 restores NMJ postsynaptic structure and improves muscle strength through normalization of muscle fiber composition and the prevention of atrophy. We hypothesize this occurs through the AChR clustering pathway in Agrn nmf380 mice. Future studies should investigate if this may represent a viable treatment option for patients with CMS, especially those with mutations in proteins of the AChR clustering pathway.

Modulation of Agrin and RhoA Pathways Ameliorates Movement Defects and Synapse Morphology in MYO9A-Depleted Zebrafish.

Congenital myasthenic syndromes (CMS) are a group of rare, inherited disorders characterised by impaired function of the neuromuscular junction (NMJ). This is due to defects in one of the many proteins associated with the NMJ. In three patients with CMS, missense mutations in a gene encoding an unconventional myosin protein, MYO9A, were identified as likely causing their disorder. Preliminary studies revealed a potential involvement of the RhoA/ROCK pathway and of a key NMJ protein, agrin, in the pathophysiology of MYO9A-depletion. In this study, a CRISPR/Cas9 approach was used to generate genetic mutants of MYO9A zebrafish orthologues, myo9aa/ab, to expand and refine the morphological analysis of the NMJ. Injection of NT1654, a synthetic agrin fragment compound, improved NMJ structure and zebrafish movement in the absence of Myo9aa/ab. In addition, treatment of zebrafish with fasudil, a ROCK inhibitor, also provided improvements to the morphology of NMJs in early development, as well as rescuing movement defects, but not to the same extent as NT1654 and not at later time points. Therefore, this study highlights a role for MYO9A at the NMJ, the first unconventional myosin motor protein associated with a neuromuscular disease, and provides a potential mechanism of action of MYO9A-pathophysiology.

The C-Terminal Fragment of Agrin (CAF), a Novel Marker of Renal Function, Is Filtered by the Kidney and Reabsorbed by the Proximal Tubule.

Agrin, a multidomain proteoglycan and neurotrypsin, a neuronal serine protease, are important for forming (neuromuscular) synapses. Proteolytical activity of neurotrypsin produces a C-terminal fragment of agrin, termed CAF, of approximately 22 kDA molecular size which also circulates in blood. The presence of CAF in urine suggests either glomerular filtration or secretion into urine. Blood levels of CAF have been identified as a potential novel marker of kidney function. Here we describe that several nephron segments in the mouse kidney express agrin and neutrotrypsin in addition to the localization of both protein in the glomerulum. Agrin mRNA and protein was detected in almost all nephron segments and mRNA abundance was highest in the inner medullary collecting duct. Neurotrypsin mRNA was mostly detected in the thick ascending limb of the loop of Henle, the distal convoluted tubule, and the inner medullary collecting duct. Moreover, we show that the proximal tubule absorbs injected recombinant CAF by a process shared with receptor-mediated and fluid phase endocytosis. Co-injection of CAF with recombinant human transferrin, a substrate of the receptor-mediated endocytic pathway as well as with FITC-labelled dextran (10 kDa), a marker of fluid phase endocytosis, showed partial colocalization of CAF with both markers. Further colocalization of CAF with the lysosomal marker cathepsin B suggested degradation of CAF by the lysosome in the proximal tubule. Thus, the murine kidney expresses agrin and neurotrypsin in nephron segments beyond the glomerulum. CAF is filtered by the glomerulum and is reabsorbed by endocytosis by the proximal tubule. Thus, impaired kidney function could impair glomerular clearance of CAF and thereby increase circulating CAF levels.