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Phosphorylation of LKB1/Par-4 establishes Schwann cell polarity to initiate and control myelin extent.

Shen YA, Chen Y, Dao DQ, Mayoral SR, Wu L, Meijer D, Ullian EM, Chan JR, Lu QR - Nat Commun (2014)

Bottom Line: The Schwann cell (SC)-axon interface represents a membrane specialization that integrates axonal signals to coordinate cytoskeletal dynamics resulting in myelination.SC-specific deletion of LKB1 significantly attenuates developmental myelination, delaying the initiation and altering the myelin extent into adulthood, resulting in a 30% reduction in the conduction velocity along the adult sciatic nerves.Phosphorylation of LKB1 by protein kinase A is essential to establish the asymmetric localization of LKB1 and Par-3 and rescues the delay in myelination observed in the SC-specific knockout of LKB1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Program in Neurosciences, University of California, San Francisco, California 94158, USA.

ABSTRACT
The Schwann cell (SC)-axon interface represents a membrane specialization that integrates axonal signals to coordinate cytoskeletal dynamics resulting in myelination. Here we show that LKB1/Par-4 is asymmetrically localized to the SC-axon interface and co-localizes with the polarity protein Par-3. Using purified SCs and myelinating cocultures, we demonstrate that localization is dependent on the phosphorylation of LKB1 at serine-431. SC-specific deletion of LKB1 significantly attenuates developmental myelination, delaying the initiation and altering the myelin extent into adulthood, resulting in a 30% reduction in the conduction velocity along the adult sciatic nerves. Phosphorylation of LKB1 by protein kinase A is essential to establish the asymmetric localization of LKB1 and Par-3 and rescues the delay in myelination observed in the SC-specific knockout of LKB1. Our findings suggest that SC polarity may coordinate multiple signalling complexes that couple SC-axon contact to the redistribution of specific membrane components necessary to initiate and control myelin extent.

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Knockout of LKB1 delays the maturation of SCs and myelination(a, b) Immunostaining for Krox20 (green) illustrates the number of promyelinating and myelinating SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. (c, d) Immunostaining for Oct6 (green) illustrates the number of premyelinating SCs. Scale bar = 100 µm. (e, f) Quantification of the number of Oct6-, Krox20-, Sox2-, S100β–positive SCs/mm2 and number of MBP-positive myelin internodes/mm2 in the WT littermate and the SC-specific LKB1 KO sciatic nerves at 14 days postnatal. Error bars represent SD. Asterisks represent significance based on Student t-test as compared to the WT control (p < 0.004). (g, h) Immunostaining for MBP (red) illustrates the myelin internodes in the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. (i, j) S100β (green) illustrates the number of SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. Nuclei are detected by DAPI (blue). Results are obtained from three knockout and three WT littermates.
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Figure 3: Knockout of LKB1 delays the maturation of SCs and myelination(a, b) Immunostaining for Krox20 (green) illustrates the number of promyelinating and myelinating SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. (c, d) Immunostaining for Oct6 (green) illustrates the number of premyelinating SCs. Scale bar = 100 µm. (e, f) Quantification of the number of Oct6-, Krox20-, Sox2-, S100β–positive SCs/mm2 and number of MBP-positive myelin internodes/mm2 in the WT littermate and the SC-specific LKB1 KO sciatic nerves at 14 days postnatal. Error bars represent SD. Asterisks represent significance based on Student t-test as compared to the WT control (p < 0.004). (g, h) Immunostaining for MBP (red) illustrates the myelin internodes in the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. (i, j) S100β (green) illustrates the number of SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. Nuclei are detected by DAPI (blue). Results are obtained from three knockout and three WT littermates.

Mentions: After the second postnatal week nerve sections were immunostained for Krox20, a transcription factor expressed specifically in myelinating SCs, as well as Oct6 and Sox2, transcription factors expressed in premyelinating and non-myelinating SCs (Figure 3a–e). Krox20 expression is robust in the myelinating SCs of the WT nerves and is significantly attenuated in the SC-specific KO nerves (Figure 3e). Whereas Oct6 and Sox2 expression is elevated in the SC-specific KO nerves as compared to WT controls (Figure 3e). The quantification of the p14 nerves suggest that the LKB1 KO SCs are held in an immature state resulting in the delayed expression of Krox20. It is important to note that the total number of SCs is unchanged along the sciatic nerves from the WT and SC-specific KO mice. Sciatic nerves were also immunostained with S100β (green) and myelin basic protein (MBP; red) to examine potential effects on SC numbers and myelination (Figure 3g–j). Myelin internodes are greatly diminished in the SC-specific KO nerves as compared to WT controls (Figure 3f–h), consistent with the luxol fast blue staining (Figure 2c). Additionally, the number of SCs is essentially unchanged upon quantification of S100β immunostaining (Figure 3f). Upon examination of SCs stained with S100β it is evident that the SCs are elongating along the axons and similar in morphology to the WT SCs (Figure 3i, j). Electron micrographs from the sciatic nerves were also examined and found to be consistent with the previous findings, that deletion of LKB1 in SCs significantly attenuates developmental myelination, delaying the initiation and altering the myelin extent into adulthood (Figure 4a). Examination of micrographs at postnatal day 14 resulted in the identification of numerous unmyelinated axons that were not yet sorted by individual SCs (Figure 4b), similar to observations in the Rac1 knockout mice17, 18. Quantification of the number of myelinated axons from the sciatic nerves, displays a significant decrease at postnatal day 7 and 14, but this difference is no longer apparent at 2 months of age (Figure 4c). From both light and electron microscopy, it is evident that myelin extent is greatly diminished in the LKB1 SC-specific KO nerves even at two months postnatal. This is clearly evident when comparing the myelin from similar sized axons from WT and LKB1 SC-specific KO nerves (Figure 4d). G-ratios display an overall increase in the SC-specific KO nerves, indicative of a decrease in the extent of myelin thickness at day 60 postnatal. These findings clearly indicate that the myelin extent from the SC-specific KO nerves remains thinner even in adulthood and does not simply represent a delay in onset of myelination (Figure 4e, Supplementary Figure 1). Together, these results suggest that LKB1 in SCs is an essential regulator for the timely initiation of myelination and controls the extent of myelination into adulthood.


Phosphorylation of LKB1/Par-4 establishes Schwann cell polarity to initiate and control myelin extent.

Shen YA, Chen Y, Dao DQ, Mayoral SR, Wu L, Meijer D, Ullian EM, Chan JR, Lu QR - Nat Commun (2014)

Knockout of LKB1 delays the maturation of SCs and myelination(a, b) Immunostaining for Krox20 (green) illustrates the number of promyelinating and myelinating SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. (c, d) Immunostaining for Oct6 (green) illustrates the number of premyelinating SCs. Scale bar = 100 µm. (e, f) Quantification of the number of Oct6-, Krox20-, Sox2-, S100β–positive SCs/mm2 and number of MBP-positive myelin internodes/mm2 in the WT littermate and the SC-specific LKB1 KO sciatic nerves at 14 days postnatal. Error bars represent SD. Asterisks represent significance based on Student t-test as compared to the WT control (p < 0.004). (g, h) Immunostaining for MBP (red) illustrates the myelin internodes in the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. (i, j) S100β (green) illustrates the number of SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. Nuclei are detected by DAPI (blue). Results are obtained from three knockout and three WT littermates.
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Figure 3: Knockout of LKB1 delays the maturation of SCs and myelination(a, b) Immunostaining for Krox20 (green) illustrates the number of promyelinating and myelinating SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. (c, d) Immunostaining for Oct6 (green) illustrates the number of premyelinating SCs. Scale bar = 100 µm. (e, f) Quantification of the number of Oct6-, Krox20-, Sox2-, S100β–positive SCs/mm2 and number of MBP-positive myelin internodes/mm2 in the WT littermate and the SC-specific LKB1 KO sciatic nerves at 14 days postnatal. Error bars represent SD. Asterisks represent significance based on Student t-test as compared to the WT control (p < 0.004). (g, h) Immunostaining for MBP (red) illustrates the myelin internodes in the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. (i, j) S100β (green) illustrates the number of SCs along the sciatic nerve of a WT littermate and the SC-specific LKB1 KO at 14 days postnatal. Scale bar = 100 µm. Nuclei are detected by DAPI (blue). Results are obtained from three knockout and three WT littermates.
Mentions: After the second postnatal week nerve sections were immunostained for Krox20, a transcription factor expressed specifically in myelinating SCs, as well as Oct6 and Sox2, transcription factors expressed in premyelinating and non-myelinating SCs (Figure 3a–e). Krox20 expression is robust in the myelinating SCs of the WT nerves and is significantly attenuated in the SC-specific KO nerves (Figure 3e). Whereas Oct6 and Sox2 expression is elevated in the SC-specific KO nerves as compared to WT controls (Figure 3e). The quantification of the p14 nerves suggest that the LKB1 KO SCs are held in an immature state resulting in the delayed expression of Krox20. It is important to note that the total number of SCs is unchanged along the sciatic nerves from the WT and SC-specific KO mice. Sciatic nerves were also immunostained with S100β (green) and myelin basic protein (MBP; red) to examine potential effects on SC numbers and myelination (Figure 3g–j). Myelin internodes are greatly diminished in the SC-specific KO nerves as compared to WT controls (Figure 3f–h), consistent with the luxol fast blue staining (Figure 2c). Additionally, the number of SCs is essentially unchanged upon quantification of S100β immunostaining (Figure 3f). Upon examination of SCs stained with S100β it is evident that the SCs are elongating along the axons and similar in morphology to the WT SCs (Figure 3i, j). Electron micrographs from the sciatic nerves were also examined and found to be consistent with the previous findings, that deletion of LKB1 in SCs significantly attenuates developmental myelination, delaying the initiation and altering the myelin extent into adulthood (Figure 4a). Examination of micrographs at postnatal day 14 resulted in the identification of numerous unmyelinated axons that were not yet sorted by individual SCs (Figure 4b), similar to observations in the Rac1 knockout mice17, 18. Quantification of the number of myelinated axons from the sciatic nerves, displays a significant decrease at postnatal day 7 and 14, but this difference is no longer apparent at 2 months of age (Figure 4c). From both light and electron microscopy, it is evident that myelin extent is greatly diminished in the LKB1 SC-specific KO nerves even at two months postnatal. This is clearly evident when comparing the myelin from similar sized axons from WT and LKB1 SC-specific KO nerves (Figure 4d). G-ratios display an overall increase in the SC-specific KO nerves, indicative of a decrease in the extent of myelin thickness at day 60 postnatal. These findings clearly indicate that the myelin extent from the SC-specific KO nerves remains thinner even in adulthood and does not simply represent a delay in onset of myelination (Figure 4e, Supplementary Figure 1). Together, these results suggest that LKB1 in SCs is an essential regulator for the timely initiation of myelination and controls the extent of myelination into adulthood.

Bottom Line: The Schwann cell (SC)-axon interface represents a membrane specialization that integrates axonal signals to coordinate cytoskeletal dynamics resulting in myelination.SC-specific deletion of LKB1 significantly attenuates developmental myelination, delaying the initiation and altering the myelin extent into adulthood, resulting in a 30% reduction in the conduction velocity along the adult sciatic nerves.Phosphorylation of LKB1 by protein kinase A is essential to establish the asymmetric localization of LKB1 and Par-3 and rescues the delay in myelination observed in the SC-specific knockout of LKB1.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Program in Neurosciences, University of California, San Francisco, California 94158, USA.

ABSTRACT
The Schwann cell (SC)-axon interface represents a membrane specialization that integrates axonal signals to coordinate cytoskeletal dynamics resulting in myelination. Here we show that LKB1/Par-4 is asymmetrically localized to the SC-axon interface and co-localizes with the polarity protein Par-3. Using purified SCs and myelinating cocultures, we demonstrate that localization is dependent on the phosphorylation of LKB1 at serine-431. SC-specific deletion of LKB1 significantly attenuates developmental myelination, delaying the initiation and altering the myelin extent into adulthood, resulting in a 30% reduction in the conduction velocity along the adult sciatic nerves. Phosphorylation of LKB1 by protein kinase A is essential to establish the asymmetric localization of LKB1 and Par-3 and rescues the delay in myelination observed in the SC-specific knockout of LKB1. Our findings suggest that SC polarity may coordinate multiple signalling complexes that couple SC-axon contact to the redistribution of specific membrane components necessary to initiate and control myelin extent.

Show MeSH