Journal of Neuroscience Research 33:205-217 (1992)

Developmental Expression of Major Myelin Protein Genes in the CNS of X-Linked Hypomyelinating Mutant Rumpshaker L.S. Mitchell, S.C. Gillespie, F. McAllister, M.L. Fanarraga, D. Kirkham, B. Kelly, P.J. Brophy, I.R. Griffiths, P. Montague, and P.G.E. Kennedy Applied Neurobiology Group (L. S.M., M.L.F., D.K., I.R.G., P.M., P.G.E.K.), and Departments of Veterinary Surgery (L.S .M., M.L.F., D .K., I.R.G., P.M.) and Neurology (P.G .E.K.), University of Glasgow, Glasgow and Department of Molecular and Biological Sciences, University of Stirling, Stirling (S.C.G., F.M., B.K., P.J.B.), Scotland

Rumpshaker (rsh) is an X-linked mutation causing hypomyelination of the CNS of mice and has recently been identified as an allele of jimpy (ip). The mutation (known asjprSh)differs in several respects from other X-linked myelin mutants, including j p , in that mice have normal longevity, oligodendrocyte numbers are not decreased, and cell death is not a feature. Myelin sheaths are deficient in immunostainable PLP protein. The present study examines the developmental expression of the major myelin protein genes and translatability of PLP and MBP mRNA. Differences between the spinal cord and brain of mutants are evident in that mRNA levels are more markedly decreased in the brain. Protein levels are severely reduced in both locations and to a proportionately greater extent than the mRNA, particularly in the spinal cord where PLP RNA and protein are approximately 80% and 10-20%, respectively, of agematched wild type mice. DM-20 protein, the other major product of the PLP gene, is disproportionately expressed in rumpshaker as is a 10 kDa proteolipid. In vitro translation studies indicate a marked decrease in PLP translation products from mutant RNA. There is no deficiency in the numbers of PLP mRNA-expressing oligodendrocytes although the abundance per cell is reduced. The data suggest that the phenotypic effects of the mutation may be associated with reduced translation of major myelin proteins, in particular PLP and its incorporation into compact myelin. However, the mutation is compatible with survival of oligodendrocytes and their differentiation to the stage of expressing PLP/DM-20 mRNA. 0 1992 Wiley-Liss, Inc. Key words: proteolipid protein, DM-20, myelin basic protein, oligodendrocyte 0 1992 Wiley-Liss, Inc.

INTRODUCTION Proteolipid protein (PLP), the major myelin protein of the CNS is a hydrophobic membrane protein (Stoffel et al., 1984; Laursen et al., 1984) postulated to be involved in formation and maintenance of the intraperiod line (Duncan et al., 1987). The PLP gene, located on the X chromosome (Willard and Riordan, 1985), has two products, PLP and DM-20, which are formed by alternative splicing of the primary transcript (Nave et al., 1987b). Both PLP and DM-20 show developmental regulation with the latter being the initial and predominant product in early development (Van Dorsselaer et al., 1987), while PLP is the major protein in mature myelin. The function of DM-20 is uncertain but based on its time of appearance and studies of certain mutations, a role in oligodendrocyte differentiation and survival has been suggested (Nadon et al., 1990; Gencic and Hudson, 1990). A possible role for DM-20 as “a mediator of adhesive membrane-membrane interactions” has also been postulated based on its surface localization in HeLa cells transfected with DM-20 cDNA (Timsit et al., 1992a). Several X-linked mutations have a severe effect on CNS myelination and in some of these a specific defect has been identified in the PLP gene. Jimpy (ip) in mice is due to a point mutation in intron 4 (Macklin et al., 1987; Nave et al., 1987a) leading to aberrant splicing (Hudson et al., 1987; Nave et al., 1986; Morello et al.,

Received March 9, 1992; revised May 24, 1992; accepted June 10, 1992. Address reprint requests to 1.R. Griffiths, Department of Veterinary Surgery, University of Glasgow, Bearsden, Glasgow G61 IQH, Scotland.

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1986) and consequent loss of exon 5 from PLP and reduced in number (Nave and Milner, 1989; CampagDM-20 transcripts. Phenotypically, hemizygous males noni and Macklin, 1988). This difference from the Xevidence tremor, seizures, and death with severe hypo- linked mutations suggests that the PLP gene may be inmyelination and reduced numbers of mature oligoden- volved in oligodendrocyte survival and as DM-20 is drocytes (Skoff, 1976). Although at 3 days of age PLP expressed prior to PLP it may have a role in cell develmRNA is 90% of control, no developmental increase opment (Nadon et al., 1990). Recently a new X-linked murine mutation, rumpoccurs and by 20 days message levels are 5% of normal (Gardinier et al., 1986) with no PLP and DM-20 proteins shaker (rsh), associated with CNS hypomyelination has detectable in the tissue (Sorg et al., 1986; Gardinier and been described (Griffiths et al., 1990). Further genetic Macklin, 1988) nor by in vitro translation (Sorg et al., analysis has indicated that this mutation is an allele of j p , 1986). Myelin basic protein (MBP) expression is also accordingly termed j f h (Cattanach and Beechey, 1991). markedly reduced though not to the same extent as for Preliminary immunocytochemical and immunoblotting PLP (Sorg et al., 1987; Gardinier et al., 1986). Jimpy data (Griffiths et al., 1990; Mitchell et al., 1990b) indimyelin synthesis deficiency GpmSd)is a phenotypically cated a severe deficiency of PLP protein in myelin but less severe allele of j p arising from a base substitution in also suggested that DM-20 was expressed in relatively exon 6 resulting in an amino acid change (Gencic and higher amounts. However, there were major differences Hudson, 1990; Macklin et al., 1991). PLP mRNA is from the previously described X-linked mutations in20-30% of normal and shows no developmental in- cluding those like jpmsd and sh-pup in which a similar crease. Reduced amounts of PLP and DM-20 protein are disproportionate expression of DM-20 occurred. jprsh is present with a disproportionate quantity of the latter not a lethal mutation and both affected hemizygous (Gardinier and M a c k h , 1988) although surprisingly the males and homozygous females can be produced; submRNA for PLP exceeds that for DM-20 (Macklin et al., stantially more myelin is present than in the other muta1991). The myelin-deficient rat (md-rut)has a base sub- tions and oligodendrocyte numbers in the spinal cord are stitution and amino acid change in exon 3 (Boison and elevated rather than reduced. Stoffel, 1989) resulting in absence of PLP and DM-20 This new mutation appears as an ideal model to protein synthesis (Yanagisawa et al., 1986; Duncan et compare with j p to determine why an apparent defect in al., 1987), severe hypomyelination (Dentinger et al., PLP expression does not result in cell death or the other 1982), and reduced oligodendrocyte numbers. The shak- severe manifestations. The PLP gene in jprshis currently ing pup (sh-pup) (Griffiths et al., 1981) also demon- being cloned but at present we do not know the molecstrates severe hypomyelination and reduced numbers of ular basis of the mutation. This paper describes the deoligodendrocytes. A base substitution in exon 2 results in velopmental profile of protein and mRNA for PLP and an amino acid change (Nadon et al., 1990) with reduced other myelin proteins in the brain and spinal cord ofjprsh expression of mRNAs for PLP (20%) and MBP (25%). relative to wild type. We find that adequate numbers of PLP and DM-20 proteins are present with a relatively PLP/DM-20 mRNA-expressing oligodendrocytes are excessive levels of DM-20 (Yanagisawa et al., 1987). present but expression of PLP protein is disproportionAll these mutations resulting from single base ately reduced compared to its message levels. This apchanges in the PLP gene affect exons coding for both pears to be due principally to impaired translation and PLP and DM-20. Their phenotypic expression has many perhaps incorporation into myelin membrane. However, common features with severe CNS hypomyelination, DM-20 and an undefined 10 kDa proteolipid are dispropaucity of oligodendrocytes, severely reduced message portionately expressed in the mutant. levels for PLP and DM-20, and reduced or absent protein. In two instances, jpmsd and the sh-pup DM-20 is present in disproportionate amounts relative to PLP. All MATERIALS AND METHODS the mutations are potentially lethal, although with careful Animals nursing the sh-pup can be reared to adulthood. One puzHemizygous (iprshlY)male and homozygous (jprsh/ zle with these mutations is why the defect in the PLP jprsh) female jprsh mice and wild-type mice from a simgene leads to reduced numbers of oligodendrocytes. In ilar genetic background (Griffiths et al., 1990) were bred j p , death of these cells occurs at the time of cell prolif- in our animal house. eration and myelination (Knapp et al., 1986; Vermeesch et al., 1990), prior to the stage of peak PLP expression. Probes for Northern Blotting and ISH The shiverer mouse has a major deletion of the MBP 32P-labeled cDNA for PLP (Milner et al., 1985), gene on chromosome 18 (Roach et al., 1985) leading to MBP (Mentaberry et al., 1986), MAG (Arquint et al., hypomyelination and the absence of MBP protein (Du- 1987) and 7 s rRNA (Balmain et al., 1982) and 35Spouey et al., 1979). However, oligodendrocytes are not labeled riboprobes were prepared exactly as described

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tion with one probe filters were stripped by incubating twice at 55°C for 30 min in a solution containing 70% formamide, 0.1 mM EDTA (pH 8.0), 10 mM tris-HC1 B (pH 7.4), and then hybridizing with a second probe. Equal amounts of total RNA (10 pg) from the brains/ spinal cords of both wild-type and mutant animals were S electrophoresed in adjacent lanes so that hybridization and exposure conditions were identical. Optical density (OD) of the autoradiographic images were measured for each sample. Equal loading of RNA between wild-type and jprsh mice at different ages was examined in both brain and B spinal cord by hybridizing with a 7 s ribosomal probe. Maximum differences over the entire age-range were less than 5%.

S PLP Fig. 1. Northern blots of total RNA (10 pg) extracted from brain (B) and spinal cord (S) and hybridized for MBP or PLP. The RNA is a pooled sample from 3 brains and 4 to 6 spinal cords at each age. The numbers above lanes indicate the age in days and are arranged so that the age lies above RNA from a wild-type mouse with the sample from the age-matched mutant on its immediate right. In general the levels of PLP and MBP are lower in the mutant but in the spinal cord the 3.2 kb transcript from jprshshows approximately the same abundance as in the wild type.

previously (Griffiths et al., 1989). The PLP probe recognizes the 5’ non-coding region and the majority of the ORF will detect both PLP and DM-20 mRNA.

Antisera A polyclonal antiserum against MBP was obtained from Dr. J-M. Matthieu. Three antisera against PLP were employed; an antiserum against the whole PLP molecule and two raised against specific peptides. One of these antipeptides recognised the PLP-specific portion of the molecule (AA 117-129) and the other was against the C-terminal common to both PLP and DM-20 (AA 271-276), (Roussel et al., 1987; Nussbaum et al., 1985). The use of the various antisera is described in “Results.” Northern and Dot Blotting Total RNA was extracted from 3 brains or 4 to 6 spinal cords from wild-type and jprsh mutant mice between the ages of 4 and 50 days. Northern and dot blotting and filter hybridizations were performed using standard techniques as described previously (McPhilemy et al., 1990; Mitchell et al., 1990a). Following hybridiza-

In Situ Hybridization (ISH) ISH was performed on 6 p m paraffin sections of spinal cord and brain from wild-type and jprsh mice between 10 and 90 days of age using a 35S-labeled RNA probe for PLP (PLP-1) which recognises the majority of the coding region and the adjacent 5‘ non-coding region (Griffiths et al., 1989). The technique used for ISH has been described fully (Griffiths et al., 1989; McPhilemy et al., 1990). The purpose of the ISH in this study was to provide an estimate of the numbers of PLP-expressing cells in the CNS. Previous quantitative studies of glial cells in jprsh (Griffiths et al., 1990) sampled the ventral columns adjacent to the ventromedian fissure of the cervical C2 segment and the mid-point of the optic nerves. These areas were again chosen for analysis using identical methods to those described for resin sections (Griffiths et al., 1990). The total number of glial cell nuclei and the proportion expressing PLP mRNA were counted in autoradiograms counterstained with haematoxylin, and the transverse areas of spinal cord and spinal white matter and optic nerve were determined. From these data the corrected total numbers of glial cells and PLP-expressing cells in transverse sections of both spinal cord white matter and optic nerve were derived. The mean values in 3 wild-type and mutant mice at each age were compared by grouped t test. (At 100 days of age only 1 optic nerve sample was available in each group.) The average signal per cell for PLP/DM-20 mRNA was compared in 16 day-old mutant and wild-type mice. Cells were enzymatically dissociated from the ventral columns of cervical cord and allowed to adhere to coverslips for 2 hr. Cells from both groups were processed in parallel to allow direct comparison. Following hybridization and development of autoradiograms the optical density of the signal from 150 cells of each group was measured and the mean values compared by t test. Full details of the dissociation, hybridization, and mea-

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Fig. 2. Developmental profiles of mRNAs for 3 myelin protein genes, PLP, MBP, and MAG, in brain and spinal cord of wild-type (m) and jpfsh(A)mice. The data are obtained from densitometric measurement of dot blots as described in “Ma-

terials and Methods.” The level of expression at 40 days of age in the wild-type mice is ascribed 100% and all other values in mutant and wild-type mice are presented relative to this.

surement techniques have been published previously (McPhilemy et al., 1990).

methionine into trichloroacetic acid (TCA)-precipitable protein (Gillespie et al., 1990).

In Vitro Translation Total RNA (100 pg/ml translation mixture; integrity of RNA was confirmed by denataturing gel electrophoresis prior to IVT) from wild-type and jprsh brains was used to program a wheat germ translation system (Roman et al., 1976) and the extent of protein synthesis was determined by measuring the incorporation of 35S-

Immunoprecipitation Immunoprecipitations were from identical numbers of TCA-precipitable radioactive protein for wild type and jprsh, 1.1 X lo6 and 2.2 X lo6 TCA-precipitable cpm for MBP and PLP, respectively. In vitro translation reaction mixes were boiled for 2 min in 2% SDS, then diluted fourfold with solution A (2.5% Triton X-100,

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Fig. 3. Developmental expression of the 3 PLP gene transcripts in mutant mouse brain and spinal cord. At each age the value for an individual transcript is presented as a percentage of the wild-type level at the same time point .( = 3.2 kb; A = 2.4 kb; 0 = 1.6 kb transcripts). The data are obtained from quantification of the northern blots after correction for any loading discrepancies (