Distal Effects of Myelination and Myelin as a Neuronal Maturation Factor

The influence of myelination is not restricted to local changes in the axon. The signaling pathways responsible for changes in the phosphorylation of cytoskeletal proteins in the axon also have other actions. Either directly or indirectly, local signals about myelination are communicated to the neuronal cell body where both translational and transcriptional changes occur. Mouse strains with altered or defective myelin provided direct evidence of this retrograde communication pathway.

A wide variety of neuronal parameters are altered in Shiverer mouse CNS tissues. These included differences in slow axonal transport, cytoskeletal composition, posttrans-lational modification of cytoskeletal proteins, and axon caliber. Even more striking than these local changes are alterations in the cytoskeletal composition of Shiverer neurons that reflect changes in neuronal gene transcription and translation. By a number of criteria, neurons in the Shiverer mouse exhibit characteristics similar to early postnatal neurons (i.e., before myelination occurs) and to neurons with nonmyelinated axons. Remarkably, analysis of these same parameters in the CNS of TG Shiverer mice producing 25% of normal myelin with minimal tremor and normal life spans reveals a phenotype exhibiting features in common with both wild-type and Shiverer neurons as well as some features intermediate between the two.

Quantitative analysis of cytoskeletal proteins in slow axonal transport provided the first evidence that the axonal cytoskeleton in a Shiverer nerve was very different from that seen in wild-type axons (Brady et al., 1999; Kirkpatrick et al., 2001). Specifically, the stoichiometries of neurofilament sub-units indicated that protein levels for high and medium molecular weight neurofilament subunits (NFH and NFM) were significantly and selectively lower in Shiverer axons (Brady et al., 1999). In contrast, NFM levels were restored to normal in TG Shiverer axons with a thin myelin sheath, but NFH was still reduced. NFL levels were not significantly different among the three mouse strains. Analysis of NF mRNA levels showed that NFH mRNA levels were reduced in both Shiverer and TG Shiverer brains, suggesting that transcription of the NFH gene was regulated by myelination (Brady et al., 1999). Because NFM mRNA levels were comparable in all three genotypes, however, control of NFM expression by myelina-tion was not regulated at the level of transcription, but rather during mRNA translation and protein expression.

Myelination levels in CNS axons dramatically affected axonal microtubules (Fig. 2). In both Shiverer and TG Shiverer axons, the number of microtubules per axon was twice that seen in comparable wild-type axons (Kirkpatrick et al., 2001). This increase was accompanied by an increase in tubulin mRNA consistent with an effect of myelin on transcription. The increased ratio of microtubules to NF and reductions in NFH/NFM levels resemble ratios obtained for the cytoskeleton of nonmyelinated axons in the CNS. These findings are consistent with the idea that formation of compact myelin sheath is a requisite step for the differentiation and maturation of large-caliber axons.

The primary phase of myelination in the rodent CNS begins 1 to 2 weeks after birth and continues for several more weeks. A number of changes in the properties of CNS neurons exhibit a similar time course. For example, NFH is low at birth and increases to approach adult levels several weeks after birth in rats (Carden et al., 1987) and levels of tubulin as well as expression of different tubulin ioforms also change during this interval (Lewis et al., 1985). Slow axonal transport is high in neonates and decreases at approximately 3 weeks postnatal (McQuarrie et al., 1989; Willard and Simon, 1983). The temporal pattern of these events, along with a failure to observe comparable changes in Shiverer, indicates that formation of compact myelination triggers these neuronal changes.

Other axonal parameters are also affected by myelination in the CNS. The density and expression levels of voltage-gated sodium channels are increased in Shiverer CNS (Noebels et al., 1991). This change reflects both a change in the distribution of the Na channels and a difference in the specific Na-channel isoforms being expressed. In nonmyelinated nerves, the predominant voltage dependent Na-channel is Na(v) 1.2. Although Na(v) 1.2 is still present in unmyelinated regions of myelinated axons, the dominant voltage dependent Na-channel in nodes of Ranvier is Na(v) 1.6 (Caldwell et al., 2000). Expression of Na(v) 1.2 is elevated in Shiverer CNS (Westenbroek et al., 1992). Moreover, Na(v) 1.2 is distributed all along the axon in Shiverer axons, much as in nonmyelinated fibers, and little

Figure 2 Distal effects of myelination on the axon cytoskeleton. Some changes in the axon reflect a change in gene transcription and protein synthesis in the neuronal cell body as a response to retrograde signals generated in the axon by myelination. Comparing the microtubule cytoskeleton of wild type axon (A) with normal myelin to a transgenic Shiverer mouse axon (B) expressing only 25% of normal myelin basic protein sufficient for a myelin sheath about 25% of wild-type; and to a Shiverer mouse axon (C) with no myelin. Both Shiverer and transgenic Shiverer axons have twice the normal number of microtubule seen in a comparable sized wild-type axon. Both Shiverer and the transgenic Shiverer nerves have increased tubulin mRNA levels as well (Kirkpatrick et al., 2001). Changes in other neuronal mRNAs including neurofilament sub-units and ion channels are also seen in Shiverer. (Figure adapted from Kirkpatrick et al., 2001.)

Figure 2 Distal effects of myelination on the axon cytoskeleton. Some changes in the axon reflect a change in gene transcription and protein synthesis in the neuronal cell body as a response to retrograde signals generated in the axon by myelination. Comparing the microtubule cytoskeleton of wild type axon (A) with normal myelin to a transgenic Shiverer mouse axon (B) expressing only 25% of normal myelin basic protein sufficient for a myelin sheath about 25% of wild-type; and to a Shiverer mouse axon (C) with no myelin. Both Shiverer and transgenic Shiverer axons have twice the normal number of microtubule seen in a comparable sized wild-type axon. Both Shiverer and the transgenic Shiverer nerves have increased tubulin mRNA levels as well (Kirkpatrick et al., 2001). Changes in other neuronal mRNAs including neurofilament sub-units and ion channels are also seen in Shiverer. (Figure adapted from Kirkpatrick et al., 2001.)

or no Na(v) 1.6 is expressed (Boiko et al., 2001). Normally Na(v) 1.2 accumulates in forming nodes of Ranvier and is then replaced by Na(v) 1.6. Concurrently, voltage-gated potassium channels (K(v) 1.1 and 1.2) appear to redistribute from a broad distribution along the nerve to a restricted distribution in juxtaparanodes as myelination proceeds (Wang et al., 1995). Expression levels and localization of K-channels are also altered in Shiverer mice with a significant increase in both neuronal and glial K-channels (Wang et al., 1995).

PNS dysmyelination mutants may also exhibit altered levels of cytoskeletal and membrane proteins. For example, MAG-deficient axons have reductions in NF protein levels that become more apparent in older axons (Yin et al., 1998). In Charcot-Marie-Tooth Type 1a disease, there appears to be reduced numbers of neurofilaments (Watson et al., 1994). Similarly, the level of specific immature ß-tubulin isotypes is elevated in CMT-1a nerves (Watson et al., 1994). Finally, K-channel expression is elevated in Trembler mouse PNS axons, as well as in Shiverer CNS axons (Wang et al., 1995).

In Trembler axons, levels of neurofilament and tubulin subunits were not dramatically altered (de Waegh et al., 1992; Kirkpatrick and Brady, 1994). However, there are a number of changes in the microtubule cytoskeleton of Trembler axons. The stable fraction of Trembler axonal microtubules is significantly reduced relative to that of wild-type axons (Kirkpatrick and Brady, 1994). This stable tubulin fraction normally increases in axons as they mature (Brady, 1984; Yan et al., 1985). The microtubule-associated protein composition in Trembler mice also is altered. Trembler axons are enriched in low-molecular-weight tau protein and the high-molecular-weight tau protein characteristic of mature peripheral nerve is reduced in Trembler relative to wild-type axons (Kirkpatrick and Brady, 1994). High-molecular-weight tau proteins result from alternative splicing of mRNAs from a single tau gene and correlate with increased maturation and MT stability (Oblinger etal., 1991).

Trembler axons are myelinated and then lose that myelin. Expression of all three NF subunits during regeneration is coordinate (McKerracher et al., 1993), but during development NFH gene expression lags behind NFL and NFM (Kost et al., 1992). This suggests that myelination of an axon may trigger a differentiation step that alters regulation of NF subunit expression. For example, MAG-null mice form myelin, but that myelin is never properly apposed to the axonal membrane, and the myelination signal may not be completed. This may explain why MAG-null mice show reductions in NF protein levels in PNS axons (Yin et al., 1998) and Trembler mice do not (de Waegh and Brady, 1991; de Waegh etal., 1992).

Dysmyelination affects neuronal gene expression in both CNS and PNS. As with the local effects of myelin, there are both similarities and differences between CNS and PNS effects of myelination. Some of these changes may reflect differences in the nature of the dysmyelination (e.g., demyelination/remyelination, failure to form compact myelin). Other differences may reflect differences in signaling between axons and OL or SC. For example, pathways expressed by SC may be split between OL and astrocytes in the CNS. Alternatively, the ability of PNS axons to regenerate more effectively than CNS fibers indicates that differences exist in the maturation of large neurons in the PNS and CNS. This may be relevant to the milder axonal pheno-type seen in many peripheral demyelinating diseases as compared to multiple sclerosis.

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