Fig. 1: A progressive demyelination and axonal degeneration affect Phb1-SCKO animals.

a Schematic representation of the floxed Phb1 allele. Exons 4 and 5 of the Phb1 gene are deleted upon Cre expression. Primers p1 and p2 were used for genotyping, while primers p1 and p3 were used to evaluate recombination. Square (exon), triangle (loxP site), blue half-arrow (primer). b Recombination PCR on DNA isolated from sciatic nerves reveals a ∼260 bp recombined band in Phb1-SCKO animals, while unrecombined DNA is too long to generate an amplicon with our PCR conditions. The experiment was repeated independently twice with identical results. c RT-qPCR analyses show a significant reduction in the level of Phb1 mRNA in sciatic nerve lysates of postnatal day 20 (P20) Phb1-SCKO mice (red) compared to controls (blue). N = 4–5 animals per genotype. Unpaired two-tailed t-test (t = 4.295, df = 7, p = 0.0036). d Representative images of cross sections of sciatic nerves. N = 3–4 animals per genotype. e The number of myelinated axons per sciatic nerve is greatly reduced in Phb1-SCKO animals starting at postnatal day 40 (P40) (middle). The decline can be explained both by demyelination (bottom) and axonal degeneration, evidenced by the reduction in the total number of axons (top). N = 3–4 animals per genotype. Unpaired two-tailed t-test corrected for multiple comparisons using the Holm-Sidak method. Total axons [P20 (t = 0.816, df = 4, p = 0.46), P40 (t = 6.508, df = 5, p = 0.0038), P60 (t = 8.08, df = 5, p = 0.0022), P90 (t = 5.257, df = 4, p = 0.012), P120 (t = 10.608, df = 4, p = 0.0022)]; myelinated axons [P20 (t = 1.053, df = 4, p = 0.35), P40 (t = 7.885, df = 5, p = 0.0016), P60 (t = 8.635, df = 5, p = 0.0014), P90 (t = 6.572, df = 4, p = 0.0055), P120 (t = 20.281, df = 4, p = 0.00017)]; amyelinated axons [P20 (t = 5.608, df = 4, 0.0099), P40 (t = 6.832, df = 5, p = 0.0041), P60 (t = 6.371, df = 5, p = 0.0042), P90 (t = 10.346, df = 4, p = 0.0024), P120 (t = 2.643, df = 4, p = 0.057)]. f Representative electron micrographs demonstrating the presence of degenerating axons (arrows), amyelinated/demyelinated axons (arrowheads) and SCs degrading their own myelin (star). N = 3 animals per genotype. g Sparse labeling of axons in the tibial nerve using the Thy1-YFP reporter mouse indicates the presence of axonal swelling at P20 (arrowheads) and axon fragmentation at P40 (arrows). N = 3–4 animals per genotype. h A functional decline is detected in Phb1-SCKO animals by electrophysiological measurements, with a reduction in nerve conduction velocity as early as P20 and decreased CMAP amplitude at P40. N = 4–8 animals per genotype. Unpaired two-tailed t-test. Velocity [P20 (t = 6.387, df = 8, p = 0.0002), P40 (t = 23.76, df = 8, p < 0.0001)]; Amplitude [P20 (t = 1.574, df = 14, p = 0.14), P40 (t = 5.635, df = 8, p = 0.0005)]. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. n.s. = non-significant.