(A) Scheme of normal (left) and impaired (right) PEX5-dependent peroxisomal protein import. PTS1, peroxisomal targeting signal type 1; PEX5, peroxisomal biogenesis factor peroxin 5. (B) Catalase (red) is present in peroxisomes (PMP70; green) of controls, but is localized in the cytoplasm of mutant fibers. PMP70, peroxisomal membrane protein 70; DAPI-stained SC nuclei are depicted in blue; Scale bar, 10 µm. (C, D) Lipid mass spectrometry of nerve lysates from controls and mutants aged 9 months indicates peroxisomal dysfunction. Peroxisomal products (PEO-) and its corresponding plasmalogens (PEP-) are reduced. Specific substrates of peroxisomal β-oxidation, VLCFA, are accumulated in mutant nerves. Statistics: means ± s.d.; n = 6; Student’s T-test; ***p<0.001. (E–G) Functional impairment of mutant compared to control sciatic nerves is assessed by electrophysiology at the age of 2 months (n = 4). To evoke significant responses of all measured nerves, larger stimulus intensity is required for mutant (0.155mA) as compared to control (0.135mA) nerves. Peak amplitudes plotted against increasing stimulus intensity indicates earlier response saturation of mutant nerves. CAP, compound action potentials; au, arbitrary units; NCV, nerve conduction velocities (each curve representing single-nerve mean responses across intensities). (H) g-ratio analysis by electron microscopy of sciatic nerves as measure of myelin thickness (n = 3; ≥100 randomly chosen axons per nerve at age 2 months). (I) Axonal analysis by methylene blue-stained semithin cross-sections of sciatic nerves of control and mutant mice at the age of 2 months (n = 3). Scale bar, 10 µm. (J) Immunostained teased sciatic nerve fibers of mutants and controls at 2 and 9 months show normal paranodal localization of CASPR (red) and anchoring protein neurofascin (NF155; green). (K, L) Intact transverse bands (arrows) at paranodal loops (PNLs) between axon (Ax) and myelin (M) by electron microscopy in a 9-month mutant nerve, even when PNLs harbor enlarged vesicles (top inset, arrowheads). Mutant nerves display normal adherens junctions (AJ) between loops (bottom inset, asterisk). Scale bars, 500 nm; scale bars in insets, 250 nm.