Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system

Abstract

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.

Figure 1. Myelination in NEX-Cre*Nrg1^flox/flox mice following embryonic recombination

(A) Neocortical development (Nissl staining) and subcortical myelination (Gallyas silver impregnation) appear normal in mutant mice (NC*F/F) compared to controls (F/F). Depicted are mirror images of coronal paraffin sections (7 µm) obtained at age 3 months. Scale bars, 1mm. (B) Top: Western blot of protein lysates revealing a loss of NRG1 in the neocortex of NC*F/F mutants compared to controls (F/+) at 3 months of age. Bottom: Densitometric quantification reveals a ~6% reduction of ‘full length’ NRG1 type III (~140 kDa) in NC*F/F mutants compared to controls (F/+). Peak intensities (±SEM) were normalized to GAPDH. (C) Semiquantitative comparison of myelination by Western blotting myelin-specific proteins from neocortical protein lysates of mutant mice (NC*F/F; age 3 months) and littermate controls (F/+). Steady state levels of CNP, MAG, MBP, and PLP/DM20 are normal. (D, E) Myelinated tracts in neocortex and corpus callosum of mutants (NC*F/F), as visualized by immunostaining for CNP. Shown are coronal paraffin sections (7 µm) of 3 months old brains from mutants (NC*F/F; right hemisphere) and control mice (F/F; left hemisphere). Scale bar, 1 mm. Enlargements (in E) reveal individual fibers in cortical layers II/III (boxed in upper panels). Scale bar, 50 µm.

Figure 2. Myelination and myelin ultrastructure in the absence of NRG1

(A) Electron microscopy reveals normally myelinated axons in the corpus callosum of mutant (NC*F/F) and control mice (wildtype and F/+) at 11 weeks of age. (B) G-ratio analysis and scatter blots derived from electron micrographs of the corpus callosum in mutant (NC*F/F) and control mice, aged 11 weeks (n=7 per genotype). Quantitation of axon size distribution reveals no obvious difference between mutants (white bars) and controls (black bars). (C) Also in aged mice (>18 months), electron microscopy of the corpus callosum demonstrates intact myelin profiles and the absence of neurodegeneration in mutant (NC*F/F) and control (F/+) mice. Scale bars, 1um. (D) Quantitation (g-ratios) of the data in (C) reveals no dys- or demyelination (n=3 per genotype). (E) Callosal myelination in the absence of NRG1 is not delayed (age P10). Confocal microscopy of coronal vibratom sections (100 µm) immunostained for axons derived from projection neurons (FNP7, red) and myelin (MBP, green) demonstrates widespread myelination in the ventral corpus callosum of mutant (NC*FF) and control (F/F) mice. Scale bar, 10 µm (inset, 250 nm). (F) Quantitation of MBP data in (E) (n=3 per genotype; ±SEM).

Figure 3. Oligodendrocytes develop on schedule in the absence of NRG1

(A) Cortical and hippocampal development in Nestin (Nes)-Cre*Nrg1F/F mice that recombine in all neural precursor cells beginning at E8.5 is without obvious delay or morphological defect (see Fig S2A). Shown are H&E stained frontal brain sections (7 µm, paraffin) of newborn controls (Nrg1F/+, top) and mutants (bottom). Scale bar, 500 µm. (B) NRG1 is virtually absent in the CNS of newborn Nes-Cre*Nrg1F/F mutant mice. Western blot analysis of protein lysates prepared from brain (top panel) and spinal cord (lower panel), comparing 3 control mice (Nrg1F/+, left) and 3 conditional null mutants (Nes-Cre*Nrg1F/F, right). Densitometry of brain and spinal cord immunoblots revealed a ~95% reduction of NRG1 in mutants compared to controls. Mean intensities (±SEM) were normalized to α-tubulin. One brain (upper lane 3) was isolated 2 hours after natural death, showing some post mortem proteolysis. Molecular weights of marker proteins are indicated (asterisks denote unspecific bands; loading control, tubulin). (C) Impaired peripheral but not central myelination. Top: Immunostaining of the ventro-medial spinal cord from newborn mice reveals the normal density of MBP+ myelin profiles (in red) in NRG1-deficient (Nes-Cre*Nrg1F/F, right) and control mice (Nrg1F/+, left). Neurons are stained for Neu-N (in green). Middle: Immunostaining of cross sections at the thoracic level for MBP (in red) and myelin protein zero (P0; in green). Note the almost complete absence of MBP and P0 in the ventral roots (VR; also marked ‘PNS’) of newborn Nes-Cre*Nrg1F/F mutants (right). In contrast, littermate Nrg1F/+ controls (left) exhibit numerous myelinated (MBP+/P0+, merged) axons. Note the presence of MBP+ oligodendrocytes in the ventro-lateral spinal chord (marked ‘CNS’) in both mutants and controls. Bottom: Immunostaining of longitudinal sections of the intercostal nerve (ICN) reveals absence of P0-stained fibers (in green) in newborn Nes-Cre*Nrg1F/F mutants (right) when compared to littermate controls (left). Scale bars, 50 µm. (D) Olig2+ oligodendrocytes (in red) are present at a normal density and with a similar distribution in the forebrain of newborn mutant mice (Nes-Cre*Nrg1F/F, right) compared to controls (Nrg1F/+, left). Boxed areas in upper panel are enlarged in lower panel. Neurons are stained for NeuN (in green). Scale bars 200 µm (upper panel), 100 µm (lower panel).

Figure 4. Myelination in the absence of ErbB3 and ErbB4 receptors

(A) Electron micrographs showing myelinated axons of the anterior corpus callosum in adult wildtype mice (labelled ‘control’) and transgenically-rescued ErbB4 single mutants (genotype MHC-ErbB4*ErbB4−/−), labeled ‘ErbB4−/−’. Scale bar, 2 µm. (B) Quantitation of myelin sheath thickness in the corpus callosum by g-ratio analysis of electron micrographs, comparing control mice (black circles, n=3) and rescued ErbB4−/− mutants (n=3, open circles). Scatter plot displays g-ratios of individual fibers as a function of the respective axon diameter. Myelin sheath thickness is not significantly different. (C) Electron micrographs of the optic nerve (top), corpus callosum (middle) and sciatic nerve (bottom) at age P11 from ErbB3*ErbB4 double mutants (right; genotype Cnp-Cre*ErbB3F/-*ErbB4F/F) and controls (genotype ErbB3F/+*ErbB4F/+). Axons in the optic nerve and corpus callosum of ErbB3*ErbB4 double mutants are normally myelinated, whereas myelination of the sciatic nerve is severly impaired. (D) Quantitation of myelin sheath thickness (g-ratios) and axon size distribution for the optic nerve of ErbB3*ErbB4 double mutants and controls (age P11; n=3 per genotype) reveals no significant difference between mutants (white circles and bars) and controls (black circles and bars).

Figure 5. Transgenic overexpression of NRG1 type I or type III causes hypermyelination

(A) Electron microscopy of hypermyelinated callosal axons in transgenic mice (age 4.5 months) overexpressing (i) NRG1 type I and (ii) NRG1 type III under control of the neuronal Thy1.2 promoter. (iii) Ultrastructure of CNS myelin and membrane spacing is indistinguishable between transgene expressing (type I tg) and wildtype (wt) axons, suggesting that hypermyelination is caused by additional membrane wraps. Scale bars, 1 µm (i; ii); 50 nm (iii). (B) CNS hypermyelination in NRG1 type I overexpressing mice. Morphometric data were obtained from 4.5 months old mice (n=3 per genotype), following electron microscopy of spinal cord (ventro-medial region, cervical segment 7) and corpus callosum (caudal region). Upper panels: when g-ratios were plotted as a function of axon size, randomly chosen fibers in spinal cord (left) and corpus callosum (right) were on avarage hypermyelinated (open rectangles, transgenic; closed rectangles, wildtype; p<0.01). Lower panels: The size distribution of axons in the same areas was not obviously inceased by neuronal NRG1 type I overexpression (white bar, transgenic; black bar, wildtype). (C) CNS hypermyelination in NRG1 type III overexpressing mice. Same analysis as in (B), with reduced g-ratios demonstrating a significant increase of myelin volume (p<0.01) in brains (left) and spinal cord (right) of Nrg1 type III transgenic mice.

Figure 6. Transgenic overexpression of NRG1 type I or NRG1 type III stimulates myelination in neocortex

(A) Quantitation of cortical hypermyelination in NRG1 type I overexpressing mice. Morphometric data were obtained from 4.5 months old animals (n=3 per genotype), following electron microscopy of neocortical layers II/III. When g-ratios were plotted as a function of axon size for randomly chosen fibers in the neocortex, hypermyelination was a feature of only the smallest (<0.4 µm) caliber axons (p<0.01), leading to crossed regression lines. (B) Cortical hypermyelination in Nrg1 type III transgenic mice. Same analysis as in (A), with reduced g-ratios demonstrating a significant increase of myelin volume (p<0.01) in brains and spinal cord Nrg1 type III transgenic mice. (C) Electron microscopy of cortical layers II/III in 4.5 months old mice (sagittal sections). Myelinated small caliber axons can be recognized (some marked by white arrowheads) in wildtype mice (i), and more numerous in Nrg1 type I transgenics (ii) and Nrg1 type III transgenics (iii). Higher magnification of boxed area (in iii) is shown on the right, depicting a normal and hypermyelinated axon (iv). Scale bars, 10 µm (i–iii) and 500 nm (iv). (D) Quantification of myelin profiles (from C) with 10 micrographs per animal analysed (n=3 per genotype; image size 440 µm²). Compared to wildtype, both NRG1 type I and NRG1 type III transgenic mice have a higher number of intracortical myelinated axons in layers II/III (p<0.01). (E) Intracortical density of CC1-stained oligodendrocytes (neocortex, layer II/III) of 4.5 months old Nrg1 type I transgenic mice (n=3) is unchanged compared to wildtype controls (n=2). Analysis of 3 images (700 µm²) per mouse.

Figure 7. Oligodendrocyte morphology

(A) Two-dimensional representations of three-dimensional tracings of CNP-stained oligodendrocytes from layers II and III of the cingulate and primary motor cortex (age 6 months). Three examples from NEX-Cre*Nrg1^flox/flox mutant (NC*F/F), control (Nrg1F/F), and Nrg1 type III-overexpressing mice (Nrg1 type III) are shown. Each color represents a primary cell process. (B-E) Quantitation of primary process number (in B), number of nodal branch points (in C), avarage process length (including internodal myelin; in D), average 3D oligodendrocyte territory (in E), and average oligodendrocyte soma volume (in F), comparing NEX-Cre*F/F (NC*F/F), Nrg1F/F and Nrg1 type III mice (12–15 cells from three mice per genotype). Error bars: SEM. Significance test: two-tailed t test with Welch's correction or Kruskal-Walli's test.

Figure 8. NRG1 type III stimulates premature myelination of the optic nerve

(A) Electron micrographs of developing optic nerves from wildtype (left) and Nrg1 type III transgenic mice (middle) at age P6, revealing single myelin profiles (arrowheads, quantified in D). Myelinated axons in boxed area (middle panel) are magnified on the right. Asterisks mark nuclei of oligodendrocytes. Scale bars, 5 µm. (B) By RT-PCR, Nrg1 type III mRNA is detectable in the eye of wildtype mice at age P1 and P30 (brain mRNA serving as internal positive controls; β-actin internal control). (C) By transgene-specific RT-PCR, type III mRNA is detectable in the eyes of postnatal (P1 and P30) Nrg1 type III trangenic mice, but not in wildtype (wt). Spinal cord mRNA (sc, age P1) as positive internal control; β-actin internal control. (D, E) Higher number of myelinated axonal profiles (in D; p<0.05) but equal number of oligodendrocytes (in E) in the optic nerve of 6 days old Nrg1 type III transgenic mice (grey bars), when compared to age-matched controls (black bars). Quantification is from semithin cross sections (wt, n=5; Nrg1 type III transgenics, n=6). Error bars, SEM (unpaired, two-sided t-test).