Figure 1. Mouse mutants with complex CNS pathology exhibit distinct but overlapping changes of myelin composition.

(A) Venn diagram summarizing CNS pathology in mice lacking the myelin proteins CNP, MAG, or PLP, according to quantitative evaluation of electron micrographs of optic nerves at P75. Note that myelin outfoldings are common to all analyzed mutants. See Figure 1—figure supplement 1A–E for quantification of these experiments. (B) Electron micrographs of P75 optic nerve cross-sections showing several myelinated axons. Myelin outfoldings and associated axons are labelled with stippled lines and asterisks, respectively. Images are representative of 4 animals per genotype, as quantified in Figure 1—figure supplement 1A. (C) Venn diagram summarizing myelin proteome alterations in Cnp^null, Mag^null, and Plp1^null-mice determined by quantitative mass spectrometry. Given are the numbers of proteins exhibiting significantly changed abundance in myelin purified from the brains of respective mutants at P75. Note that several septins are diminished in all analyzed mutants. n=3 animals per genotype. See Figure 1—source data 1 for entire dataset and Figure 1—figure supplement 1G–I for validation by immunoblot. (D) Immunolabelling validates diminishment of SEPT8 (green) in myelinated fibre tracts of Cnp^null, Mag^null, and Plp1^null-mice. Longitudinally sectioned optic nerves of P75 mice are shown. The axonal marker TUJ1 (red) was co-labelled as a control. Images are representative of three independent experiments.

DOI: http://dx.doi.org/10.7554/eLife.17119.003

Figure 1—figure supplement 1. Neuropathological and molecular analysis in mouse models of complex CNS pathology (Cnp^null, Mag^null, and Plp1^null mice).

(A–E) Quantification of CNS pathology as summarized in main Figure 1A. Electron micrographs of optic nerves were evaluated for the indicated pathologies. Note that myelin outfoldings are common to all three mutants. n=4 mice per genotype; age P75; one-way ANOVA with Bonferroni’s multiple comparisons test comparing to WT. p-values are as follows: (A) WT vs. Cnp^null p=0.002, WT vs. Mag^null p=0.02, WT vs. Plp1^null p=0.002; (B) WT vs. Cnp^null p=0.11, WT vs. Mag^null p=0.02, WT vs. Plp1^null p<0.001; (C) WT vs. Cnp^nullp<0.001, WT vs. Mag^null p<0.001, WT vs. Plp1^null p=0.56; (D) WT vs. Cnp^null p<0.001, WT vs. Mag^null p>0.999, WT vs. Plp1^null p=0.37; (E) WT vs. Cnp^null p=0.22, WT vs. Mag^nullp>0.999, WT vs. Plp1^nullp=0.0012. (F) Diminishment of myelin septins in models of myelin-related pathology according to label-free proteomics. Myelin was purified from the brains of the indicated mutants and littermate controls, and analyzed by quantitative mass spectrometry. Given is the abundance in myelin of SEPT2, SEPT4, SEPT7, SEPT8 relative to their respective littermate controls. Note that the abundance in myelin of SEPT4, SEPT7, and SEPT8, was significantly reduced in all analyzed mutants. The diminishment in myelin of SEPT2 was significant in Cnp^nulland Plp1^nullmice and at the border of significance in Mag^nullmice. See Figure 1—source data 1 for the entire dataset. n=3 mice per genotype; age P75; unpaired two-tailed t-test; p-values are as follows: SEPT2: p=0.012 (Cnp^null), p=0.0614 (Mag^null), p=0.013 (Plp1^null); SEPT4: p<0.001 (Cnp^null), p=0.002 (Mag^null), p<0.001 (Plp1^null); SEPT7: p<0.001 (Cnp^null), p=0.0114 (Mag^null), p=0.009 (Plp1^null); SEPT8: p=0.007 (Cnp^null) p=0.011 (Mag^null) p=0.011 (Plp1^null). (G–I) Immunoblotting validates the diminished abundance of septins (SEPT2, SEPT4, SEPT7, SEPT8) in CNS myelin of all three models. Genotypes and equal loading were controlled by immunoblotting. Blots are representative of 3 animals per genotype. (J–M) Abundance of septin-mRNAs in models of myelin-related pathology. The abundance of the indicated mRNAs in a myelin-rich white matter tract (corpus callosum) of the indicated models was determined by qRT-PCR. The abundance of Sept2, Sept7, and Sept8 mRNA was unaltered in all models (J, L, M). The abundance of Sept4 was moderately reduced in Cnp^null and Mag^null corpus callosi (K). Corpus callosi of n=4 animals per genotype were analyzed. One-way ANOVA with Bonferroni’s multiple comparison test comparing to WT. (J) WT vs. Cnp^null p=0.0767, WT vs. Mag^null p=0.3661, WT vs. Plp1^null p> 0.9999; (K) WT vs. Cnp^null p=0.006, WT vs. Mag^null p=0.0014, WT vs. Plp1^null p=0.175; (L) WT vs. Cnp^null p=0.45, WT vs. Mag^null, p>0.999, WT vs. Plp1^null p=0.085; (M) WT vs. Cnp^null p>0.999, WT vs. Mag^null p>0.999, WT vs. Plp1^null p=0.14.

DOI: http://dx.doi.org/10.7554/eLife.17119.005