Evidence against altered forms of MAG in the dysmyelinated mouse mutant claw paw

In the mammalian nervous system, the formation of myelin byglial cells enables a rapid saltatory impulse conduction. Myelina-tion depends on the coordinated expression of myelin-specificstructural proteins, based on interactions of axons with oligoden-drocytes in the central nervous system (CNS) and Schwann cells inthe peripheral nervous system (PNS). Mice with defects in myelinassembly comprise a large and genetically heterogeneous group ofspontaneous mutants (Nave 1994), which also serve as models forhuman neurological diseases (Snipes and Suter 1995). Several my-elin-deficient mice carry defects in genes yet unidentified.The murine autosomal mutation claw paw (genotype clp/clp)produces abnormalities of limb posture associated with a myelina-tion disorder of the peripheral nervous system, but not the centralnervous system (Henry et al. 1991). Peripheral nerve myelinationis greatly delayed, and the failure to myelinate results in the per-sistence of promyelin Schwann cell/axon units, involving predomi-nantly small-caliber fibers. The clinical abnormalities, which areobvious within 2 days after birth, include abnormally flexed andextended joints, most obvious in the forelimbs. In human, congen-ital joint contractures (arthrogryposis) may be a presenting featureof congenital hypomyelinating neuropathy (Boylan et al. 1992)and have been associated with mutations in the gene for the majorperipheral myelin protein P0 (Warner et al. 1996). Unlike patientswith arthrogryposis, however, clp/clp mice display nonfixed ab-normal limb postures in early life. Very severely affected mutantsdie within 2–3 days after birth, but those that survive the firstseveral weeks have a normal life span. The claw paw gene, whichis molecularly undefined, has been mapped by linkage analysisclose to the glucose phosphate isomerase-1 locus (Gpi-1)onmouse Chromosome (Chr) 7 (Henry et al. 1991).Myelin-associated glycoprotein (MAG) is a myelin-specificcell adhesion molecule of the immunoglobulin super-family ofmembrane proteins (Arquint et al. 1987; Lai et al. 1987). Themurine Mag gene has been mapped to mouse Chr (Barton et al.1987) where it is also closely linked to the Gpi-1locus. The MAGprotein is a minor constituent of CNS and PNS myelin, and itsdistribution at the glial-axonal interface suggests a specific role inthe early steps of axonal recognition and myelin formation.In myelinating glia, two MAG isoforms (termed S-MAG andL-MAG) are generated by alternative RNA splicing of exon 12,which carries an in-frame termination codon (Lai et al. 1987). Theresulting MAG-mRNAs encode a 67-kDa (S-MAG) and a 72-kDaisoform (L-MAG), which differ by the length of their carboxyltermini (Milner et al. 1990). In the rodent brain, these two MAGisoforms are developmentally regulated, with L-MAG being themajor neonatal form. In the peripheral nervous system, S-MAG isthe major form at all developmental stages.In an attempt to demonstrate the function of MAG in vivo, anull allele of the Mag gene has been created by gene targeting(Montag et al. 1994; Li et al. 1994). Surprisingly, MAG-deficientmouse mutants displayed no obvious developmental defects, ex-cept for subtle ultrastructural abnormalities of myelin, in markedcontrast to the dysmyelinated phenotype of claw paw, which isthus highly unlikely to be a functional Mag null allele. It remainsunknown which myelin proteins are able to compensate for theabsence of MAG in this null allele. In contrast, transgenic over-expression of the murine Mag gene may not be tolerated (M.Klugmann, A. Schneider, D. Montag, and K.-A. Nave, unpub-lished observation). However, even in mice with 8–10 transgeniccopies of the wild-type MAG gene, neurological deficits are rela-tively subtle and the clinical phenotype is transient and differentfrom that of claw paw mice. Nevertheless, MAG overexpression inmice suggested to us that alterations of this protein may result inaberrant gain-of-function effects. A well-documented example thatmissense mutations can underlie a severe dysmyelination (whereasthe complete deletion of the same gene is compatible with myelinassembly) is provided by the proteolipid protein (PLP) gene, en-coding the major component of CNS myelin (Klugmann et al.1997). Given the close linkage between Mag and clp (see below),Mag qualified as a candidate gene for the claw paw mutation:alterations of the MAG coding sequence could, for example, exerta misfolded protein with dominant-negative effects on other mem-brane proteins, yet without having the full penetrance of an auto-somal-dominant mutation. In fact, heterozygous claw paw miceare viable, but some myelin abnormalities have been described(Henry et al. 1991).A search of all linkage data available from the Mouse GenomeDatabase (Jackson Laboratory), comprising a total of 332 mice,did not show any recombination between Mag and Gpi-1(Rudert1992; Saunders 1990; Tong 1993; Fig. 1A). Since clp is alsoclosely linked to Gpi-1(Henry et al. 1991), the physical proximityof Mag and clp can be inferred. To test directly the possible in-volvement of MAG in the claw paw defect, we determined thecDNA-sequence of both MAG-isoforms in mice homozygous forthe clp mutation. Assuming that peripheral and central MAG pro-teins are molecularly indistinguishable, cDNA was reverse-transcribed from total brain RNA of a visibly affected mutant andan age-matched wild-type control. In independent amplifications,which followed standard procedures, three overlapping fragmentsof the MAG cDNA were generated, including 47 bp at the 58 endand 14 bp at the 38 end of the open reading frame (Fujita et al.1989), with different primer pairs corresponding to the knownexon sequences (Fig 1B). The PCR-generated products had theexpected sizes of 433 bp (with primer pair E3-AS6), 394 bp(primer pair S6-AS8), and 980 bp (primer pairS8-E13). RT-PCR