Formation of compact myelin is required for maturation of the axonal cytoskeleton

Abstract

Although traditional roles ascribed to myelinating glial cells are structural and supportive, the importance of compact myelin for proper functioning of the nervous system can be inferred from mutations in myelin proteins and neuropathologies associated with loss of myelin. Myelinating Schwann cells are known to affect local properties of peripheral axons (de Waegh et al., 1992), but little is known about effects of oligodendrocytes on CNS axons. The shiverer mutant mouse has a deletion in the myelin basic protein gene that eliminates compact myelin in the CNS. In shiverer mice, both local axonal features like phosphorylation of cytoskeletal proteins and neuronal perikaryon functions like cytoskeletal gene expression are altered. This leads to changes in the organization and composition of the axonal cytoskeleton in shiverer unmyelinated axons relative to age-matched wild-type myelinated fibers, although connectivity and patterns of neuronal activity are comparable. Remarkably, transgenic shiverer mice with thin myelin sheaths display an intermediate phenotype indicating that CNS neurons are sensitive to myelin sheath thickness. These results indicate that formation of a normal compact myelin sheath is required for normal maturation of the neuronal cytoskeleton in large CNS neurons.

Fig. 1.

SCa and SCb rates are increased in shiverer relative to control and MBP/MBP shiverer transgenic rates.A, Slow axonal transport rates were examined by segmental analysis. [³⁵S]methionine was injected into the vitreous of the mouse eye, and 21 d after injection, optic nerve and tracts were harvested and cut into 1 mm segments. Radioactively labeled proteins in each segment were resolved on SDS-PAGE gels, processed for fluorography, and exposed to film. Fluorographs of control, MBP/MBP shiverer transgenic, and shiverer animals show the wave of radioactively labeled SCa proteins traveling down the optic nerve and tract. The positions for neurofilament subunits (H, M, and L) and tubulin doublet (T) are distributed as a wave in all three animals 21 d after injection. Note that the peaks for NFH and NFM are in axon segments 3–4 mm for control and MBP/MBP nerves, but are in segments 5–6 mm for shiverer. Similarly, the peak of tubulin is 4 mm in control and MBP/MBP shiverer transgenic animals but 5.5 mm in shiverer animals. Note that NFH is barely detectable in shiverer and MBP/MBP animals when compared to control, but NFL levels are comparable. B, Distance from retina for labeled peaks of actin at 4, 5, and 7 (actin) and for NFM at 18, 21, and 24 d after labeling. Data are plotted for shiverer (triangles), MBP/MBP transgenics (circles), and wild-type control (filled squares), where each symbol is data from a different animal, and two to five animals were used for each time point. A best fit line passing through zero is shown for shiverer (short dashed line, triangle), MBP/MBP transgenic (long dashed line, circle), and wild-type control (solid line, filled square). The slope of this line represents the rate of slow transport.

Fig. 2.

Axonal neurofilament and microtubule organization and numbers are altered in MBP/MBP shiverer transgenic and shiverer optic axons relative to control. Typical axonal cytoskeletons are shown in electron micrographs of optic nerve axonal cross sections from wild-type (A, B), MBP/MBP transgenic (C, D), and shiverer (E, F) mice. The number and density of microtubules (arrow) are significantly increased in MBP/MBP transgenic and shiverer axons relative to wild-type control axons. Neurofilament organization (arrowhead) appears altered to produce increased density, although these changes are less dramatic than those seen with microtubules. Neurofilament numbers may be depressed in the shiverer and MBP/MBP transgenic axons. Compare the completely myelinated axons in control to the partially myelinated axons of MBP/MBP shiverer transgenic animals and unmyelinated axons of shiverer. Although the density of extracellular material appears similar in micrographs from each category of mouse, the electron density of axoplasm appears greatest in MBP/MBP transgenics and is also increased in shiverer axons. Scale bar, 0.19 μm.

Fig. 3.

Morphometric analyses show changes for both microtubule and neurofilament density in shiverer and MBP/MBP shiverer transgenic axons. As a measure of cytoskeletal organization, the density of microtubules and neurofilaments was examined as described previously (de Waegh et al., 1992). The number of cytoskeletal elements in random hexagons was evaluated. Both microtubules and neurofilament densities are shifted to higher values in shiverer axons.A, Microtubule distributions were similar in shiverer and MBP/MBP transgenic nerves. The mean number of microtubules per hexagon was increased in both MBP/MBP transgenic (4.38 ± 0.05; ± SEM) and shiverer (4.53 ± 0.05; ± SEM) axons. These differences were significant at p ≤ 0.0001 (two-samplet test) when compared to the wild-type control value of 1.9 ± 0.07 (± SEM) MT per hexagon. B, Unlike microtubules, the neurofilament distribution in MBP/MBP transgenic nerves was intermediate between shiverer and wild-type control nerves. The mean number of neurofilaments per hexagon was also significantly greater in shiverer (4.39 ± 0.06; ± SEM neurofilaments per hexagon) and MBP/MBP shiverer transgenic (4.75 ± 0.05; ± SEM neurofilaments per hexagon; in a paired t test relative to wild-type control) than in control (4.11 ± 0.05 neurofilaments per hexagon). Differences relative to wild-type control axons are significant at p ≤ 0.0001 (shiverer) andp = 0.002 (MBP/MBP transgenic) in a pairedt test. Morphometric analyses were conducted by overlaying a hexagonal grid over electron micrographs of the optic axonal cross sections printed at a final magnification of 140,000×. Each hexagon represented an area of 0.035 μm². The number of microtubules and neurofilaments per hexagon was scored, binned, and plotted. The number of microtubules per hexagon ranged between 0 and 12, whereas the number of neurofilaments per hexagon ranged from 0 to 23.

Fig. 4.

Neurofilament protein levels and neurofilament composition are changed in MBP/MBP shiverer transgenic and shiverer animals relative to control. The amount of NFH, NFM, and NFL protein in nerves expressed as a fraction of the total protein moving with SCa was determined by densitometry of fluorographs from axonal transport studies in wild-type control, MBP/MBP transgenic, and shiverer mice.A, NFH protein levels are decreased relative to wild-type control in both MBP/MBP transgenic and shiverer (n = 17; n = 24; andn = 17, respectively). The differences in NFH for shiverer was different from wild-type at p = 0.051, whereas MBP/MBP transgenics were different at p = 0.037. Shiverer and transgenics had a similar mean value for NFH, but the shiverer data had greater scatter. NFM levels were decreased in shiverer mice relative to both wild-type (p= 0.035) and transgenics (p ≤ 0.0001), but NFM in MBP/MBP transgenic animals was increased over wild-type (p = 0.04). NFL levels were not distinguishable between wild-type control and MBP/MBP transgenics, but were increased slightly in shiverer relative to both wild-type control (p = 0.026) and MBP/MBP transgenics (difference significant at p ≤ 0.0001). All comparisons were made using a two-sample t test.B, Changes in the relative contributions of NFH, NFM, and NFL proteins to neurofilament mass illustrate changes in composition. A larger proportion of shiverer neurofilament consists of NFL subunits, whereas in control animals the contribution of NFH to the neurofilament mass is greater than either MBP/MBP transgenic or shiverer neurofilaments. Similarly, in MBP/MBP shiverer transgenic the contribution of NFH to neurofilament mass is comparable to that in shiverer, but the NFM content is greater than in either control or shiverer animals.

Fig. 5.

Expression levels of NFH are significantly reduced in shiverer as opposed to wild-type control. The reduced levels of NFH polypeptide in shiverer and MBP/MBP transgenic is reflected in reduced levels of NFH mRNA. In contrast, NFM mRNA is not significantly different from wild-type control in either shiverer or MBP/MBP transgenic animals. A, Northern blot of NFH and the GAPDH loading control. The expression of the 4.0 kb NFH transcript appeared to be reduced in shiverer when compared to control or MBP/MBP transgenic animals. The NFH mRNA levels in MBP/MBP transgenic appeared to be slightly reduced. Each lane contains 5 μg of total RNA isolated from the cortices of age-matched animals. B,Quantitation of the NFH expression levels (n = 5). After normalizing with GAPDH, a ratio of NFH expression was calculated, and the expression level of shiverer and MBP/MBP shiverer transgenic was normalized to control expression levels. The levels of NFH expression were significantly reduced in shiverer relative to control (p = 0.029 in a paired ttest). Although levels of NFH expression in MBP/MBP shiverer transgenic animals were consistently reduced as opposed to wild-type control and consistently higher than shiverer NFH expression levels, the differences were not significant at p ≥ 0.05.C, Northern blot of NFM and GAPDH loading control. No consistent differences in mRNA expression levels were apparent.D, Quantitation of relative NFM levels in the three animals (n = 11). NFM levels were not significantly different in the three animal types.

Fig. 6.

Quantitative immunoblot analysis of highly phosphorylated NFH antibody immunoreactivity in corpus callosum and sciatic nerve homogenates from MBP/MBP shiverer transgenic and shiverer relative to wild-type control. The relative phosphorylation of NFH was significantly reduced in CNS, but not in PNS axons. Whereas NFH levels were reduced in the CNS of MBP/MBP transgenic and shiverer mice, the phosphorylation of the NFH present was roughly half that of wild-type controls. The differences from wild-type control values were significant at p = 016 for shiverer and atp = 0.0014 for MBP/MBP transgenics. Phosphorylation of NFH was not significantly different between MBP/MBP transgenic and shiverer nerves. Approximately 10 μg of corpus callosum or sciatic nerve homogenates was run on SDS-PAGE gels and transferred to nitrocellulose blots, then blotted with the RMO24 antibody, which is specific to highly phosphorylated NFH. This was followed by a radioactively labeled secondary antibody before being exposed to a PhosphorImager screen. The relative intensity of highly phosphorylated NFH immunoreactivity on the ensuing scans was determined. Highly phosphorylated NFH signals were normalized to NFL-immunoreactive signals that had been corrected for varying levels among the three animal types using the data in Figure 4. Each was plotted as the ratio of NFH-immunoreactive signal from MBP/MBP or shiverer to control. All values were corrected for the varying amounts of NFH protein levels among the three animal types using the data in Figure 4 before calculation of phosphorylation ratios. For corpus callosum samples,n = 32 for shiverer animals, n= 21 for MBP/MBP animals, and n = 17 for control animals. For sciatic nerve samples, n = 32 for shiverer, n = 20 for MBP/MBP, andn = 18 for control animals.

Fig. 7.

Pathways for compact myelin to affect CNS neuronal differentiation. Although the molecular identity of many components involved in the modulation of neuronal architecture by myelinating oligodendrocytes remains to be determined, essential characteristics of these pathways can be defined. In this diagram, some of these key elements are illustrated schematically. One or more signals is produced by interaction between compact myelin and axon (small arrows). These signals are not produced by simple interaction between an oligodendrocyte process and the axon, such as is seen in shiverer (arrows in circle withbar), but require formation of compact myelin. Signals produced in axon segments surrounded by compact myelin suggest multiple actions. The first may be an action on local factor or factors (eight-pointed star) that changes the net activity of axonal kinases and/or phosphatases, thereby altering the net phosphorylation state of neurofilaments in that segment, much as was seen in Trembler PNS neurons. In this figure, the open symbols represent factors in the absence of myelin, whereas thefilled symbols correspond to factors that have been “activated” in the presence of compact myelin. A second action might involve a retrograde acting factor (filled star) that is committed to retrograde axonal transport and returned to the cell body. At the level of the cell body, this retrograde signal may activate other pathways, directly alter transcription in the nucleus (stars innucleus), or act at the level of translation in the cell body (stars near polysomes). In the absence of appropriate myelination, axonal signals might fail to be activated (unfilled stars in axon), leading to an altered composition and organization of the axonal cytoskeleton, or may not be returned to the cell body.