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. Author manuscript; available in PMC: 2019 Sep 4.
Published in final edited form as: Cell Metab. 2018 Jul 12;28(3):400–414.e8. doi: 10.1016/j.cmet.2018.06.011

Figure 6. Calpastatin is necessary for Mfn2-mediated protection of neuromuscular synapses.

Figure 6

(A) Representative immunoblot and quantification of calpastatin in spinal cords of mice injected with AAV1-shCon. or shRNA targeting calpastatin (AAV1-shCalpastatin) into lumbar spinal cords of 4–6 month old TMFN mice for 2 weeks. n = 3 mice per group. (B) Quantification of NMJ innervation in transected or sham-operated GAST muscles of 4–6 month old TMFN mice 2 days after right sciatic nerve cut. 12 days before transection, mice were injected with indicated AAV1 into lumbar spinal cords. n = 3 mice per group. (C and D) Age of paralysis onset (C, n = 5–8 mice per group) and quantification of NMJ innervation (D, n = 5, 4, 3, 5 mice per group, 90-day old or paralyzed mice) in G93A and dTg mice injected with indicated AAV1 into lumbar spinal cords at 60-day old. (E) Schematic of generation of Rosa26-hCAST KI mice. Upon Cre recombination, STOP cassette is removed, resulting in human calpastatin (CAST) expression under CMV early enhancer/chicken beta actin (CAG) promoter. Representative western blot detection of human calpastatin and Mfn2 in spinal cords of TCAST mice shown in the right panel (note that all used commercially available calpastatin antibodies have much higher affinity to human calpastatin than mouse calpastatin. The immunoblot analysis does not reflect the actual level of human calpastatin overexpression). (F and G) Quantification of NMJ innervation (F) and representative immunoblot and quantification of βIII tubulin, NF-L and the calpain cleaved 150-kDa αII-spectrin breakdown product (SBDP150) in sciatic nerves (G) of 4–6 month old TCAST mice 2 days after right sciatic nerve cut. n = 3 mice per group. (H) Hypothetical model of Mfn2-mediated protective effects on neuromuscular synapses. First, calpastatin forms the complex on MAMs. Then, likely through the intermolecular interaction between MAM-Mfn2 and mitochondrial Mfn2, calpastatin-containing MAMs are connected to mitochondria and further transport along axons by moving mitochondria. The delivery of calpastatin to distal nerve endings leads to the localized inhibition of calpain, which finally prevents axon degeneration and neuromuscular synaptic loss. Equal protein amounts of 20 μg were loaded for all blots. Data are means ± s.e.m., representative of triplicate experiments. One-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.0001, ****P < 0.0001. ns, not significant.