Figure 5.

SNX17 knockdown decreased LRP4 expression and MuSK phosphorylation in the gastrocnemius muscle of EAMG mice and aggravated the degree of AChR fragmentation at the NMJ along with leg weakness. (A) Small-animal live imaging of EAMG mice infected with AAV2/9-EGFP NC and AAV2/9-m-SNX17 shRNA-EGFP for 21 days in the gastrocnemius muscle of the right leg. (B) Western blotting and qPCR analyses of SNX17 protein (n = 3/group) and mRNA (n = 9/group), respectively, in the mouse gastrocnemius muscle tissue. (C) Western blotting analyses of LRP4, MuSK, and p-MuSK levels in the mouse gastrocnemius muscle tissue. n = 3/group. (D) AChR (red) at the NMJ of the gastrocnemius filaments was labeled with R-BTX and the area of AChRs was quantified with ImageJ. n = 6/group; Scale bar = 50 μm. (E) Hematoxylin and eosin–stained pathological analyses of cross-sections of the gastrocnemius muscle fibers of mice in each group. Black arrows indicate muscle fiber atrophy, rupture, or inflammatory cell infiltration. Scale bar = 200 μm. (F) EMG of the gastrocnemius muscle was measured using low-frequency RNS. The attenuation rate of the fifth amplitude of EMG compared with the first amplitude was calculated. SNX17 KD-con: n = 3; SNX17 KD: n = 5. (G) Bar dynamometer measurement of the right leg strength of mice in each group. NC: n = 6; SC: n = 5; SNX17 KD-con: n = 5; SNX17 KD: n = 8. All data are presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001. AChR, acetylcholine receptor; EAMG, experimental autoimmune myasthenia gravis; LRP4, low-density lipoprotein receptor-related protein 4; MuSK, muscle-specific receptor tyrosine kinase; NC, normal control; NMJ, neuromuscular junction; R-BTX, rhodamine-labeled bungarotoxin; RNS, repetitive nerve stimulation; SC, solvent control; SEM, Standard Error of Mean; SNX17, sorting nexin 17. ns, no significance.