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The raft-associated protein MAL is required for maintenance of proper axon--glia interactions in the central nervous system.

Schaeren-Wiemers N, Bonnet A, Erb M, Erne B, Bartsch U, Kern F, Mantei N, Sherman D, Suter U - J. Cell Biol. (2004)

Bottom Line: These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered.Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts.Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology, Department of Research, University Hospital Basel, 4056 Basel, Switzerland. Nicole.Schaeren-Wiemers@unibas.ch

ABSTRACT
The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode--axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

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Alterations in myelin and raft component incorporation in mal KO mice. (A) Western blot analysis of myelin proteins from 4-mo-old WT and KO mice. Brain myelin from three animals of each genotype. Note besides the absence of MAL in KO mice, reduced protein levels of L-MAG, MBP, NF155, and Caspr in the mutants. (B) Western blot analysis of myelin DIG preparations. CHAPS extraction and sucrose step gradient ultracentrifugation from myelin preparations (12 collected gradient fractions). Fraction 1–5: low density (5% sucrose, not depicted); fraction 6: 5%/30% sucrose transition zone with the floating DIGs, fractions 7–10 contain ∼30% sucrose (7–8 not depicted), and fraction 11 and 12 contain the detergent-soluble proteins in 40% sucrose. Quantification of myelin proteins in myelin (C) and myelin DIG (D) preparations from KO mice. The relative protein levels in brain myelin (A) and in CHAPS-insoluble fraction 6 (B) were quantified. Values express the percentage of protein in KO compared with WT samples (*, P < 0.05). (E) Western blots of L-MAG, Caspr, and both NF isoforms 155 and 186 in myelin and plasma membrane preparations of 4-mo-old WT mouse brain. (F) CHAPS extraction and sucrose step gradient ultracentrifugation from myelin and plasma membranes of WT mice. (G) CHAPS extraction and sucrose step gradient ultracentrifugation from brain myelin preparation of P9 WT mice. In contrast to contactin/F3, L-MAG and NF155 are localized in the soluble fractions. Note that at this developmental stage the extent of myelination differs significantly between brain regions; small amounts of MAL were detectable in the lower brainstem.
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fig8: Alterations in myelin and raft component incorporation in mal KO mice. (A) Western blot analysis of myelin proteins from 4-mo-old WT and KO mice. Brain myelin from three animals of each genotype. Note besides the absence of MAL in KO mice, reduced protein levels of L-MAG, MBP, NF155, and Caspr in the mutants. (B) Western blot analysis of myelin DIG preparations. CHAPS extraction and sucrose step gradient ultracentrifugation from myelin preparations (12 collected gradient fractions). Fraction 1–5: low density (5% sucrose, not depicted); fraction 6: 5%/30% sucrose transition zone with the floating DIGs, fractions 7–10 contain ∼30% sucrose (7–8 not depicted), and fraction 11 and 12 contain the detergent-soluble proteins in 40% sucrose. Quantification of myelin proteins in myelin (C) and myelin DIG (D) preparations from KO mice. The relative protein levels in brain myelin (A) and in CHAPS-insoluble fraction 6 (B) were quantified. Values express the percentage of protein in KO compared with WT samples (*, P < 0.05). (E) Western blots of L-MAG, Caspr, and both NF isoforms 155 and 186 in myelin and plasma membrane preparations of 4-mo-old WT mouse brain. (F) CHAPS extraction and sucrose step gradient ultracentrifugation from myelin and plasma membranes of WT mice. (G) CHAPS extraction and sucrose step gradient ultracentrifugation from brain myelin preparation of P9 WT mice. In contrast to contactin/F3, L-MAG and NF155 are localized in the soluble fractions. Note that at this developmental stage the extent of myelination differs significantly between brain regions; small amounts of MAL were detectable in the lower brainstem.

Mentions: To search for an explanation of the phenotype observed in myelinated CNS axons of mal-deficient mice, and based on the hypothesis that the function of MAL as a component of rafts in regulating protein sorting and trafficking might be affected, we quantified the levels of myelin proteins in myelin and in myelin raft preparations of KO and WT animals (Fig. 8). Myelin from brains of 4-mo-old mice (n = 3) was analyzed by Western blot analysis (Fig. 8 A). We compared the relative protein levels in WT and KO animals for the compact myelin proteins PLP/DM20 and myelin basic protein (MBP), the two noncompact myelin proteins myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), and the transmembrane protein NF155 and its binding partner contactin/F3 and Caspr (Fig. 8 C). MAG levels, the 72-kD isoform of MAG (L-MAG), and MBP were significantly reduced to ∼60% in myelin derived from KO mice compared with WT samples (Fig. 8 C). As in our confocal analyses, NF155 protein levels were reduced in myelin membranes from KO animals (Fig. 8 A), and Caspr and contactin/F3 were also consistently reduced to ∼80% (Fig. 8, A and C). In contrast, the amount of PLP/DM20 in myelin membranes of KO animals was slightly increased, whereas MOG incorporation into KO myelin was not affected (Fig. 8 C).


The raft-associated protein MAL is required for maintenance of proper axon--glia interactions in the central nervous system.

Schaeren-Wiemers N, Bonnet A, Erb M, Erne B, Bartsch U, Kern F, Mantei N, Sherman D, Suter U - J. Cell Biol. (2004)

Alterations in myelin and raft component incorporation in mal KO mice. (A) Western blot analysis of myelin proteins from 4-mo-old WT and KO mice. Brain myelin from three animals of each genotype. Note besides the absence of MAL in KO mice, reduced protein levels of L-MAG, MBP, NF155, and Caspr in the mutants. (B) Western blot analysis of myelin DIG preparations. CHAPS extraction and sucrose step gradient ultracentrifugation from myelin preparations (12 collected gradient fractions). Fraction 1–5: low density (5% sucrose, not depicted); fraction 6: 5%/30% sucrose transition zone with the floating DIGs, fractions 7–10 contain ∼30% sucrose (7–8 not depicted), and fraction 11 and 12 contain the detergent-soluble proteins in 40% sucrose. Quantification of myelin proteins in myelin (C) and myelin DIG (D) preparations from KO mice. The relative protein levels in brain myelin (A) and in CHAPS-insoluble fraction 6 (B) were quantified. Values express the percentage of protein in KO compared with WT samples (*, P < 0.05). (E) Western blots of L-MAG, Caspr, and both NF isoforms 155 and 186 in myelin and plasma membrane preparations of 4-mo-old WT mouse brain. (F) CHAPS extraction and sucrose step gradient ultracentrifugation from myelin and plasma membranes of WT mice. (G) CHAPS extraction and sucrose step gradient ultracentrifugation from brain myelin preparation of P9 WT mice. In contrast to contactin/F3, L-MAG and NF155 are localized in the soluble fractions. Note that at this developmental stage the extent of myelination differs significantly between brain regions; small amounts of MAL were detectable in the lower brainstem.
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Related In: Results  -  Collection

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fig8: Alterations in myelin and raft component incorporation in mal KO mice. (A) Western blot analysis of myelin proteins from 4-mo-old WT and KO mice. Brain myelin from three animals of each genotype. Note besides the absence of MAL in KO mice, reduced protein levels of L-MAG, MBP, NF155, and Caspr in the mutants. (B) Western blot analysis of myelin DIG preparations. CHAPS extraction and sucrose step gradient ultracentrifugation from myelin preparations (12 collected gradient fractions). Fraction 1–5: low density (5% sucrose, not depicted); fraction 6: 5%/30% sucrose transition zone with the floating DIGs, fractions 7–10 contain ∼30% sucrose (7–8 not depicted), and fraction 11 and 12 contain the detergent-soluble proteins in 40% sucrose. Quantification of myelin proteins in myelin (C) and myelin DIG (D) preparations from KO mice. The relative protein levels in brain myelin (A) and in CHAPS-insoluble fraction 6 (B) were quantified. Values express the percentage of protein in KO compared with WT samples (*, P < 0.05). (E) Western blots of L-MAG, Caspr, and both NF isoforms 155 and 186 in myelin and plasma membrane preparations of 4-mo-old WT mouse brain. (F) CHAPS extraction and sucrose step gradient ultracentrifugation from myelin and plasma membranes of WT mice. (G) CHAPS extraction and sucrose step gradient ultracentrifugation from brain myelin preparation of P9 WT mice. In contrast to contactin/F3, L-MAG and NF155 are localized in the soluble fractions. Note that at this developmental stage the extent of myelination differs significantly between brain regions; small amounts of MAL were detectable in the lower brainstem.
Mentions: To search for an explanation of the phenotype observed in myelinated CNS axons of mal-deficient mice, and based on the hypothesis that the function of MAL as a component of rafts in regulating protein sorting and trafficking might be affected, we quantified the levels of myelin proteins in myelin and in myelin raft preparations of KO and WT animals (Fig. 8). Myelin from brains of 4-mo-old mice (n = 3) was analyzed by Western blot analysis (Fig. 8 A). We compared the relative protein levels in WT and KO animals for the compact myelin proteins PLP/DM20 and myelin basic protein (MBP), the two noncompact myelin proteins myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG), and the transmembrane protein NF155 and its binding partner contactin/F3 and Caspr (Fig. 8 C). MAG levels, the 72-kD isoform of MAG (L-MAG), and MBP were significantly reduced to ∼60% in myelin derived from KO mice compared with WT samples (Fig. 8 C). As in our confocal analyses, NF155 protein levels were reduced in myelin membranes from KO animals (Fig. 8 A), and Caspr and contactin/F3 were also consistently reduced to ∼80% (Fig. 8, A and C). In contrast, the amount of PLP/DM20 in myelin membranes of KO animals was slightly increased, whereas MOG incorporation into KO myelin was not affected (Fig. 8 C).

Bottom Line: These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered.Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts.Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

View Article: PubMed Central - PubMed

Affiliation: Neurobiology, Department of Research, University Hospital Basel, 4056 Basel, Switzerland. Nicole.Schaeren-Wiemers@unibas.ch

ABSTRACT
The myelin and lymphocyte protein (MAL) is a tetraspan raft-associated proteolipid predominantly expressed by oligodendrocytes and Schwann cells. We show that genetic ablation of mal resulted in cytoplasmic inclusions within compact myelin, paranodal loops that are everted away from the axon, and disorganized transverse bands at the paranode--axon interface in the adult central nervous system. These structural changes were accompanied by a marked reduction of contactin-associated protein/paranodin, neurofascin 155 (NF155), and the potassium channel Kv1.2, whereas nodal clusters of sodium channels were unaltered. Initial formation of paranodal regions appeared normal, but abnormalities became detectable when MAL started to be expressed. Biochemical analysis revealed reduced myelin-associated glycoprotein, myelin basic protein, and NF155 protein levels in myelin and myelin-derived rafts. Our results demonstrate a critical role for MAL in the maintenance of central nervous system paranodes, likely by controlling the trafficking and/or sorting of NF155 and other membrane components in oligodendrocytes.

Show MeSH
Related in: MedlinePlus