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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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Targeted disruption of the mal locus and generation of a mal KO mouse line. (A) Partial restriction map of the mal locus (top), and schematic representation of the targeting construct (middle) and the targeted locus (bottom). Homologous recombination results in the concomitant deletion of exon 1 (Ex1) and insertion of a LacZ/Neo cassette that follows an artificially generated NotI site after the 3rd amino acid of the mal ORF in the targeting construct. HSV-Tk: herpes simplex virus thymidine kinase gene. E, X, S, and K: EcoRV, XbaI, SacII, and KpnI, respectively. Bar, 1 kb. Inset: Southern blot analysis of DNA isolated from tail biopsies, digested with EcoRV. The blot was probed with a radioactively labeled XbaI–XbaI fragment (indicated in A as 5′ probe). A 3.4-kb fragment indicates a mutant allele (−/−), whereas a 5.8-kb fragment represents a WT allele (+/+). (B) Immunohistochemistry for MAL on tissue sections from sciatic nerve and optic tract of 3-mo-old WT (top panels) and KO (bottom panels) mice. Arrowheads demarcate the border between optic tract and brain. Bars, 100 μm. (C) Western blot analysis of myelin membranes from 3-mo-old WT and KO mice using antibodies to MAL (left) and MOG (right). (D) β-Galactosidase activity, detected by X-Gal staining, in homozygous mice. Bars, 100 μm.
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fig1: Targeted disruption of the mal locus and generation of a mal KO mouse line. (A) Partial restriction map of the mal locus (top), and schematic representation of the targeting construct (middle) and the targeted locus (bottom). Homologous recombination results in the concomitant deletion of exon 1 (Ex1) and insertion of a LacZ/Neo cassette that follows an artificially generated NotI site after the 3rd amino acid of the mal ORF in the targeting construct. HSV-Tk: herpes simplex virus thymidine kinase gene. E, X, S, and K: EcoRV, XbaI, SacII, and KpnI, respectively. Bar, 1 kb. Inset: Southern blot analysis of DNA isolated from tail biopsies, digested with EcoRV. The blot was probed with a radioactively labeled XbaI–XbaI fragment (indicated in A as 5′ probe). A 3.4-kb fragment indicates a mutant allele (−/−), whereas a 5.8-kb fragment represents a WT allele (+/+). (B) Immunohistochemistry for MAL on tissue sections from sciatic nerve and optic tract of 3-mo-old WT (top panels) and KO (bottom panels) mice. Arrowheads demarcate the border between optic tract and brain. Bars, 100 μm. (C) Western blot analysis of myelin membranes from 3-mo-old WT and KO mice using antibodies to MAL (left) and MOG (right). (D) β-Galactosidase activity, detected by X-Gal staining, in homozygous mice. Bars, 100 μm.

Mentions: We used embryonic stem cell technology to generate mal-deficient mice by replacing the first exon of the mal gene with a lacZ gene (Fig. 1 A). Southern blot analysis of tail genomic DNA yielded a 5.8-kb EcoRV fragment for the wild-type (WT) allele and a diagnostic 3.4-kb EcoRV fragment for the knockout (KO) allele (Fig. 1 A, inset). Northern blot analysis of brain RNA revealed that the expression of full-length MAL mRNA was abolished in homozygous mice, whereas the LacZ-containing transcript was expressed in both heterozygous and homozygous mutant mice (unpublished data). Lack of MAL protein was shown by immunohistochemistry with an affinity-purified polyclonal anti-MAL rabbit serum on sections from sciatic nerves and brain of 3-mo-old KO animals (Fig. 1 B). We found strong MAL expression in myelin of sciatic nerve and optic tract from WT mice (Fig. 1 B, top). In contrast, MAL immunoreactivity was abolished in KO animals (Fig. 1 B, bottom). Absence of MAL was further confirmed by Western blot analysis of myelin preparations from WT and KO mice (Fig. 1 C). X-gal staining revealed β-galactosidase activity in sciatic and optic nerves of heterozygous and homozygous mice (Fig. 1 D).


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)

Targeted disruption of the mal locus and generation of a mal KO mouse line. (A) Partial restriction map of the mal locus (top), and schematic representation of the targeting construct (middle) and the targeted locus (bottom). Homologous recombination results in the concomitant deletion of exon 1 (Ex1) and insertion of a LacZ/Neo cassette that follows an artificially generated NotI site after the 3rd amino acid of the mal ORF in the targeting construct. HSV-Tk: herpes simplex virus thymidine kinase gene. E, X, S, and K: EcoRV, XbaI, SacII, and KpnI, respectively. Bar, 1 kb. Inset: Southern blot analysis of DNA isolated from tail biopsies, digested with EcoRV. The blot was probed with a radioactively labeled XbaI–XbaI fragment (indicated in A as 5′ probe). A 3.4-kb fragment indicates a mutant allele (−/−), whereas a 5.8-kb fragment represents a WT allele (+/+). (B) Immunohistochemistry for MAL on tissue sections from sciatic nerve and optic tract of 3-mo-old WT (top panels) and KO (bottom panels) mice. Arrowheads demarcate the border between optic tract and brain. Bars, 100 μm. (C) Western blot analysis of myelin membranes from 3-mo-old WT and KO mice using antibodies to MAL (left) and MOG (right). (D) β-Galactosidase activity, detected by X-Gal staining, in homozygous mice. Bars, 100 μm.
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Related In: Results  -  Collection

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fig1: Targeted disruption of the mal locus and generation of a mal KO mouse line. (A) Partial restriction map of the mal locus (top), and schematic representation of the targeting construct (middle) and the targeted locus (bottom). Homologous recombination results in the concomitant deletion of exon 1 (Ex1) and insertion of a LacZ/Neo cassette that follows an artificially generated NotI site after the 3rd amino acid of the mal ORF in the targeting construct. HSV-Tk: herpes simplex virus thymidine kinase gene. E, X, S, and K: EcoRV, XbaI, SacII, and KpnI, respectively. Bar, 1 kb. Inset: Southern blot analysis of DNA isolated from tail biopsies, digested with EcoRV. The blot was probed with a radioactively labeled XbaI–XbaI fragment (indicated in A as 5′ probe). A 3.4-kb fragment indicates a mutant allele (−/−), whereas a 5.8-kb fragment represents a WT allele (+/+). (B) Immunohistochemistry for MAL on tissue sections from sciatic nerve and optic tract of 3-mo-old WT (top panels) and KO (bottom panels) mice. Arrowheads demarcate the border between optic tract and brain. Bars, 100 μm. (C) Western blot analysis of myelin membranes from 3-mo-old WT and KO mice using antibodies to MAL (left) and MOG (right). (D) β-Galactosidase activity, detected by X-Gal staining, in homozygous mice. Bars, 100 μm.
Mentions: We used embryonic stem cell technology to generate mal-deficient mice by replacing the first exon of the mal gene with a lacZ gene (Fig. 1 A). Southern blot analysis of tail genomic DNA yielded a 5.8-kb EcoRV fragment for the wild-type (WT) allele and a diagnostic 3.4-kb EcoRV fragment for the knockout (KO) allele (Fig. 1 A, inset). Northern blot analysis of brain RNA revealed that the expression of full-length MAL mRNA was abolished in homozygous mice, whereas the LacZ-containing transcript was expressed in both heterozygous and homozygous mutant mice (unpublished data). Lack of MAL protein was shown by immunohistochemistry with an affinity-purified polyclonal anti-MAL rabbit serum on sections from sciatic nerves and brain of 3-mo-old KO animals (Fig. 1 B). We found strong MAL expression in myelin of sciatic nerve and optic tract from WT mice (Fig. 1 B, top). In contrast, MAL immunoreactivity was abolished in KO animals (Fig. 1 B, bottom). Absence of MAL was further confirmed by Western blot analysis of myelin preparations from WT and KO mice (Fig. 1 C). X-gal staining revealed β-galactosidase activity in sciatic and optic nerves of heterozygous and homozygous mice (Fig. 1 D).

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