Histopathological Findings and Expression of NEP
An immunohistochemical assay with an anti‐NEP/CD10 antibody showed expression of NEP in the germinal center of human tonsil tissue, in which NEP is highly expressed on proliferating B cells, confirming the specificity of the antibody against NEP (Fig 6A). Using this antibody, we confirmed the obvious expression of NEP in the myelin sheath of the sural nerve from the control patient, although it was also slightly expressed in the axon (Fig 6B). We obtained sural nerve biopsies from P1 and P4. Both showed a remarkable decrease in the density of large myelinated fibers with thin myelin sheaths and clusters of myelinated fibers (Fig 6C,E). Inflammatory cells and onion‐bulb formation were absent. Immunohistochemical staining with an anti‐NEP/CD10 antibody showed a negative and a partial positive reaction in P1 and P4, respectively, compared to myelin of the sural nerve from the control patient (Fig 6D,F). In addition, the antibody detected a band migrating at approximately 90kDa, corresponding to NEP, in the homogenates from the sural nerve of the control patient by western blotting, but the band was not detected in P1 (Fig 6G).
Figure 6  Neprilysin immunohistochemistry and western blot analysis. (A) Immunohistochemical staining with an anti‐NEP/CD10 antibody revealed the expression of NEP/CD10 (brown) in the marginal center of human tonsil. (B) Expression of NEP/CD10 (brown) was observed in myelin and axons, especially at the outer surface of myelin of the sural nerve from a control patient (inset). (C and E) Toluidine blue staining of a sural nerve. Densities of large myelinated fibers are markedly decreased in both patient 1 (C) and 4 (E), who harbor the c.654+1G>A (p.Gly179fs) and c.1861T>C (p.Cys621Arg) mutation, respectively. Clusters of small myelinated fibers are occasionally noted. (D and F) Expression of NEP/CD10 (brown) was not detected in the nerve from P1 (D), but partially detected in the nerve from P4 (F). (G) Western blot analysis of NEP/CD10. Blotted bands were detected with a rabbit anti‐NEP/CD10 antibody (arrowhead). NEP/CD10 was detected in homogenates from the sural nerve of the control patient, but not P1. White bar, 100 μm; black bar, 20 μm. CP = control patient; MW = molecular weight marker; P1 = patient 1; Ton = human tonsil.

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