Myelin Structural Integrity in a Model for Human Early-Onset CMT1B

Abstract

Charcot-Marie-Tooth disease type 1B (CMT1B), a peripheral neuropathy, is caused by mutations in MPZ, the gene encoding protein zero (P0), the major integral protein of PNS myelin. An adhesive protein, P0 plays a significant role during elaboration and maintenance of multilamellar myelin. P0 mutation Arg69Cys (R69C) causes a severe early-onset form of CMT1B. To elucidate the pathogenesis of this neuropathy, an Arg69Cys knock-in mouse was generated by targeting the Arg69Cys mutation to one MPZ allele by homologous recombination in ES cells. Here we report our x-ray diffraction (XRD) measurements on the periodicity, membrane structure, and amount of myelin in unfixed, freshly-dissected nerves from wildtype (WT or +/+), heterozygous (R69C/+), and homozygous (R69C/ R69C) mice. CNS myelin (optic nerve) was also examined. The diffraction patterns showed decreasing strength of scattering intensity from myelin: WT > R69C/+ > R69C/R69C, indicating decreasing relative amounts of myelin. By contrast, optic nerves exhibited no such differences among genotypes. From the positions of the reflections the myelin periods of sciatic but not optic nerves were found to differ among the genotypes: 177.0 ± 0.4 A for WT, 178.4 ± 0.5 A for R69C/+, and 193.1 ± 4.2 A for R69C/R69C. The calculated electron density profiles showed R69C/R69C's wider period derived from ∼20 A-swelling at the extracellular apposition. The extent of membrane packing distortion (Δ/d) in PNS myelin, calculated using Bragg order peak widths, was 25% greater in R69C/+ and doubled in R69C/R69C compared to WT. Differences in amount of myelin, period, and Δ/d among the genotypes were statistically significant at p < 0.001. Finally, comparison of R69C/+ with P0± and R69C/R69C with P0-/- suggested the small amount of mutant P0 that enters the myelin may detrimentally affect myelin-myelin interactions to produce less regular/unstable packing.