Study of the Interaction of the Myelin Monomeric GTP-Binding Proteins with Other Brain Proteins

Abstract: Although several monomeric GTP-binding proteins have been found in myelin, the signaling pathways in which they operate are not known. To define these signaling pathways we searched for specific target proteins that interact with the myelin monomeric GTP-binding proteins. A blot overlay approach was used. Bovine white matter homogenate, myelin, and oligodendrocyte proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose membranes. The presence of proteins that interact with the myelin GTP-binding proteins was explored by incubating those blots with an enriched fraction of 22- and 25-kDa myelin GTP-binding proteins labeled with radioactive guanine nucleotides. When the GTP-binding proteins were in the inactive state (GDP-bound) they interacted with 28-, 47-, and 58-kDa oligodendrocyte polypeptides. Only the 28-kDa protein was present in myelin. In the active state (GTP-bound), they interacted only with a 47-kDa protein in myelin but with 31-, 38-, 47-, 58-, 60-, 68-, and 71-kDa proteins in oligodendrocytes and total homogenate. Under these experimental conditions the 28-kDa protein did not interact with the GTP-binding proteins. The fact that the myelin GTP-binding proteins in the active state formed complexes with a different set of proteins than when in the inactive state is a strong indication that these proteins are effector proteins. With the exception of the 31- and 38-kDa proteins that were detected only in the cytoplasmic fraction, these polypeptides were detected in the cytosolic fraction and total membrane fraction. The 25-kDa GTP-binding protein was present in all the complexes. Immunoblot analysis indicated that the 28-kDa polypeptide is RhoGDI, an effector protein that is known to regulate the activation and movement of several GTP-binding proteins between different cellular compartments. Thus, this study opens the way to identify the macromolecules participating in the myelin signaling pathway involving monomeric GTP-binding proteins.     

Hydrolysis of Inositol Trisphosphate by Purified Rat Brain Myelin

Abstract: Highly purified rat brain myelin was found to hydrolyze inositol 1,4,5-trisphosphate to inositol 1.4-bisphosphate, but subsequent hydrolysis of the latter, characteristic of whole brainstem, did not occur. Inositol 1,4,5-trisphosphate 5-phosphatase in myelin was ∼ 33% of the level in microsomes and 127% that of the cytosolic fraction from brainstem. The myelin and microsomal enzymes had similar properties, as follows: activation by saponin, requirement for Mg²⁺ and similar K_act (0.16 and 0.13 mM), K_m (8.7 ± 2.5 and 7.0 ± 1.0 μM), and pH optima (6.6-6.8). V_max values were 11.2 ± 1.0 and 26.3 ± 2.0 nmol/mg/min for myelin and microsomes, respectively. A possible role for this enzyme in phosphoinositide-mediated signal transduction within myelin and its subcompartments is discussed.     

Phospholipid Biosynthesis in Myelin: Presence of CTP: Phosphoethanolamine Cytidylyltransferase in Purified Myelin of Rat Brain

Highly purified myelin from rat brain was previously shown to contain the ethanolaminephosphotransferase which completes the synthesis of phosphatidyl ethanolamine. We have now obtained evidence for the presence in myelin of CTP:phosphoethanolamine cytidylyltransferase, the enzyme catalyzing formation of CDP-ethanolamine. Myelin was isolated by two different procedures, one based on the Norton-Poduslo method and the other involving repetitive gradients with osmotic shocking deferred to the end. The fact that activity remained constant through all but the earliest steps suggested that the enzyme is intrinsic to myelin. Comparison of subcellular fractions revealed that approximately half the total activity was in the supernatant, the remainder being distributed among the particulate fractions. Relative specific activity of myelin was 27-31% that of microsomes, thus eliminating the possibility of appreciable contamination by the latter. The possibility of adsorption of the soluble enzyme by myelin was rendered unlikely by retention of activity after washing the myelin with buffered sodium chloride or sodium taurocholate. Furthermore, relative specific activity of the cytidylyltransferase was 10-fold higher than that of lactate dehydrogenase (a cytosolic marker) in myelin. The apparent Km for CTP was approximately the same for myelin and microsomes, but that for phosphoethanolamine was significantly higher for myelin.     

Ethanolamine Kinase Activity in Purified Myelin of Rat Brain

Highly purified rat brain myelin showed a significant level of ethanolamine kinase, amounting to 17% of the specific activity of whole brain homogenate. This kinase level in myelin was an order of magnitude higher than that of lactate dehydrogenase, a marker for cytosol. Subcellular distribution studies revealed that in addition to myelin, this kinase was present in the P1, P2, P3, and cytosolic fractions with highest relative specific activity in the latter. The possibility that myelin activity resulted from adsorption of the soluble enzyme was unlikely since activity was retained in myelin that had been washed with buffered sodium chloride or taurocholate. Mixing experiments and repeated purification further indicated that the enzyme is intrinsic to myelin. Kinetic studies indicated similar Km values for ethanolamine in the microsomal, cytosolic, and myelin fractions but a significantly lower apparent Km for ATP in myelin. This and other differences suggested the possible existence of isozymes. Establishment of the presence of this kinase completes the list of phospholipid synthesizing enzymes needed to synthesize phosphatidylethanolamine from diacylglycerol within the myelin membrane.     

Interactions of Muscarinic Receptors with the Heterotrimeric G Proteins Gq and G12: Transduction of Proliferative Signals

Abstract: The proliferative and transforming properties of m2 and m5 muscarinic acetylcholine receptors and a series of wild-type, chimeric, and mutant G proteins were measured alone or in combination in NIH 3T3 cells to determine which G proteins mediate these signals and to what extent these signals can be influenced by changing the stoichiometry of receptors and G proteins. Responses were measured using the focus-forming assay and a novel assay called R-SAT (R eceptor S election and A mplification T echnology) in which proliferative responses are monitored using a reporter gene. Individually, GTPase-deficient mutants (^?) of Gαq and Gα12, wild-type Gαq, and m5 were active in R-SAT. Gα12^? and m5 also induced focus formation. m2 was inactive in both assays. The ability of m5 to induce foci was significantly reduced by coexpression of Gαq^?. Synergistic effects of receptor/G protein combinations were not observed in focus-forming assays but were readily detected by R-SAT. Coexpression of Gαq with m5 induced constitutive activity in R-SAT and increased the potency of agonists at m5 by 90-fold. Gαq also evoked agonist-dependent responses from m2 but not constitutive activity. Agonist potency was increased 10-fold at m2 and decreased 15-fold at m5 when these receptors were coexpressed with Gαqi5, a chimeric G protein containing the five C-terminal residues of Gαi2, compared with coexpression with Gαq. Both Gαq and Gαqi5 had biphasic effects on the proliferative responses to m5 and m2, respectively, inhibiting responses at high agonist concentrations. Coexpression of Gα12 or Gα12i5 had no effect on the concentration-response relationships of m5, but both elicited weak responses from m2. We conclude that although Gα12 is a more potent oncogene, Gαq transduces m5-driven cellular responses. The demonstrations that proliferative responses can be elicited from a nonmitogenic receptor by altering the type and concentration of available G proteins and that constitutive responses can be induced by G proteins imply that both the magnitude and type of receptor-initiated signal can be regulated at the level of G proteins in vivo.     

5''-Nucleotidase in Rat Brain Myelin

Rat brain myelin showed substantial activity of 5'-nucleotidase. The specific activity in myelin was enriched two- to threefold over that in rat brain homogenates, and the total activity in myelin accounted for approximately 24% of the activity in the homogenates. The 5'-nucleotidase in the homogenates and in isolated myelin had optimum activity at pH 7.5--9.0, was stimulated by Mg2+ and Mn2+, and was inhibited by Co2+, Zn2+, EDTA, and EGTA. 5'-AMP, 5'-UMP, and 5'-CMP were the preferred substrates, and 5'-GMP was hydrolyzed at approximately one-half the rate of the other mononucleotides. The very low rates of cleavage of beta-glycerophosphate and 2'-AMP ruled out any significant contribution of nonspecific phosphatase to the observed 5'-nucleotidase activity in myelin. The 5'-nucleotidase was inhibited by concanavalin A and was protected by alpha-methyl-D-mannoside against inhibited by that lectin, suggesting that this enzyme in the CNS is a glycoprotein. It is concluded from these data, and from histochemical observations made in other laboratories, that the myelin sheath is one major locus of 5'-nucleotidase in the rat brain.     

Biochemical Study of Heterosis for Brain Myelin Content in Mice

Heterosis (hybrid vigor) for brain myelin content has been examined in detail in (C57BL/6J x DBA/2J)F1 hybrid mice at 17 days of age. The amount of myelin isolated from the F1 hybrid brain is greater than that isolated from either parental strain. In addition, the total protein content in the myelin of the three genotypes showed the following trend: F1 greater than DBA greater than C57. However, no discernible differences in myelin protein compositions could be detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the whole brain for several myelin-associated constituents such as GM1 ganglioside, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase), 5'-nucleotidase, and carbonic anhydrase indicated that heterosis exists for these components. No heterosis was found for such nonmyelin constituents as gangliosides GD1a, GT, GQ, RNA, DNA, and choline acetyltransferase. A developmental study of the whole brain CNPase indicated that the heterotic effect was greatest during the most active period of myelination (17-30 days). We conclude that the heterotic effect is specific for myelin content and is probably the result of an accelerated myelin synthesis. The heterotic effect should have great potential as a new model for studying aspects of myelinogenesis.     

Activation of Complement by Myelin: Identification of C1-Binding Proteins of Human Myelin from Central Nervous Tissue

Myelin isolated from central nervous tissue activates the classic pathway of complement by directly activating C1. Activation of C1 can proceed to form membrane attack complex, C5b-9, in the myelin. Such an interaction between myelin and complement may be important in diseases involving myelin damage, in view of the role of complement in membrane attack and inflammation. To identify the C1-activating protein, myelin was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. The blots were incubated with C1 or with whole serum complement, followed by immunostaining for C1 or C3, respectively. A duplicate strip was stained with amido black or anti-myelin antibody to visualize the myelin proteins. The results showed that two major protein bands were capable of activating C1. An approximately 56-58-kilodalton band comigrated with the W2 protein and an approximately 45-47-kilodalton band migrated along with, but slightly behind, the W1 Wolfgram doublet.     

Quantitative Changes in Myelin Proteins in a Peripheral Neuropathy Caused by Tullidora (Karwinskia humboldtiana)

Peripheral nerve demyelination was induced in cats by oral administration of ether extracts of Tullidora (Karwinskia humboldtiana). Proteins from several hindlimb nerves, spinal roots, and dorsal columns of the spinal cord were subjected to slab gel electrophoresis and quantified by densitometry. In Tullidora-treated cats with severe motor disturbances, specific myelin proteins were reduced by at least 50% in motor nerves and less than 25% in cutaneous axons. There was a greater decrease of these proteins in the distal than in the cephalad segments of the sciatic nerve; no changes were detected either in the spinal roots or in the white matter of the spinal cord. Electron microscopy revealed intense demyelination in the motor nerves only. Both the density of the 100 A-thick neurofilaments and the relative proportion of a polypeptide with a molecular weight of 68,000 were considerably increased in the affected nerves. It is tentatively concluded that the active principles of Tullidora may enter the axons through the motor nerve terminals. The distal segments of the motor nerves would then be preferentially affected and demyelination could result from axonal damage.     

Immunoblot Identification of 13.5 Kilodalton Myelin Basic Protein in Goldfish Brain Myelin

Myelin isolated from goldfish brain shows a multilamellar structure with a major dense line and two intraperiod lines. Sodium dodecyl sulfate gel electrophoresis revealed that the protein profile of goldfish brain myelin is distinctly different from that of rat brain myelin. No protein migrating to the position of proteolipid protein or DM-20 was seen in goldfish myelin. Goldfish acclimated to 5 degrees, 15 degrees, and 30 degrees C showed no qualitative differences in myelin proteins. The 13.5 kD protein in goldfish brain myelin and brain homogenate was intensely immunostained with the antiserum to human basic protein by the immunoblot technique. In contrast, none of the proteins of goldfish myelin were immunostained with antiproteolipid protein serum; however, both proteolipid protein and DM-20 of rat brain myelin were immunostained. The significance of the synthesis of myelin proteins by astrocytes in the goldfish brain is discussed.     

Studies on the Wolfgram High Molecular Weight CNS Myelin Proteins: Relationship to 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase

Evidence is presented that the major protein components of the high molecular weight CNS myelin proteins designated as the Wolfgram protein doublet (W1 and W2) contain the enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (EC 3.1.4.37, CNP). CNP is a basic hydrophobic protein containing about 830 to 840 amino acid residues. When electrophoresed on SDS polyacrylamide gels, CNP appears as a protein doublet, separated by a molecular weight difference of about 2500-3000 in bovine, human, rat, guinea pig, and rabbit. A similar protein doublet has been identified as the Wolfgram proteins W2 and W1 in myelin and in the chloroform-methanol-insoluble pellet obtained from myelin. Moreover, the relative Coomassie blue staining intensity of the CNP2 plus CNP1 protein doublet among the species examined was remarkably similar to that observed for electrophoresed myelin and chloroform-methanol-insoluble pellet derived from myelin. Antisera raised against purified bovine CNP recognized the W1 and W2 proteins isolated from bovine and human brain. The amino acid composition of pure bovine CNP is presented and compared with the compositions of several rat and bovine Wolfgram proteins obtained by other investigators. Our electrophoretic, compositional, and immunological data support the contention that the enzyme CNP is a major component of the Wolfgram protein doublet.     

Pertussis toxin reverses Gpp(NH)p inhibition of basal and forskolin activated adipocyte adenylate cyclase

Inhibition of basal adenylate cyclase by GTP or guanyl-5'-yl imidodiphosphate was abolished in membranes isolated from rat adipocytes previously incubated with pertussis toxin. Forskolin (0.1 microM) stimulated adenylate cyclase about 4-fold and inhibition of cyclase by GTP or guanyl-5'-yl imidodiphosphate was also abolished by pertussis toxin treatment of rat adipocytes. Forskolin (1 microM) increased adenylate cyclase activity at least ten-fold and the inhibitory effect of GppNHp was reduced but not abolished by pertussis toxin. In rabbit adipocytes, pertussis toxin reversed the inhibition of adenylate cyclase activity by GppNHp to the same extent as that by GTP in the presence of 1 microM forskolin. The present results indicate that pertussis toxin can reverse the inhibition of adipocyte adenylate cyclase by nonhydrolyzable GTP analogs as well as that by GTP.     

Complement Regulatory Molecules on Human Myelin and Glial Cells: Differential Expression Affects the Deposition of Activated Complement Proteins

Abstract: The expression of decay-accelerating factor CD55, membrane cofactor protein CD46, and CD59 was studied on Schwann cells cultured from human sural nerve and myelin membranes prepared from human cauda equina and spinal cord. These proteins are regulatory membrane molecules of the complement system. CD55 and CD46 are inhibitors of C3 and C5 convertases and CD59 inhibits C8 and C9 incorporation into C5b-9 complex and C9-C9 polymerization. The presence of these proteins was assessed by using antibodies to each of the proteins by fluorescent microscopy, fluorescence-activated cell sorter analysis, and also sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Schwann cells in culture expressed CD55, CD46, and CD59. It is interesting that only CD59 was detected on myelin from both central and peripheral nerve tissue. The ability of these proteins to limit C3 peptide deposition and C9 polymerization in myelin was studied by western blot analysis. C3b deposition was readily detected on antibody-sensitized myelin incubated with normal human serum used as a source of complement but not with EDTA-treated or heat-inactivated serum. C3b deposition was not affected by anti-CD55 antibody. On the other hand, poly-C9 formation in myelin, which was maximum when 50% normal human serum was used, was increased four- to fivefold when myelin was preincubated with anti-CD59. Our data suggest that complement activation on myelin is down-regulated at the step of the assembly of terminal complement complexes, including C5b-9, due to the presence of CD59.     

Myelin-Deficient Rat: Analysis of Myelin Proteins

Myelin basic protein (BP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), and 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) activity were quantitated in the brains and spinal cords of normal and myelin-deficient (md) rats at 8, 12, 18, and 25 days of age. The levels of BP, MAG, and CNP in 25-day-old md brain were 1.1, 1.8, and 11% of those in controls, respectively. In spinal cord, the levels were higher, at 9, 15, and 12% of control values, respectively. Although BP content in the mutant rats was a lower percentage of the control level than MAG and CNPase contents at all ages, the absolute level of BP increased steadily between 8 and 25 days of age in both brain and spinal cord, whereas there was little change in the amounts of MAG and CNPase during this period. Immunoblotting analysis did not reveal an increased apparent Mr for MAG, as has been observed in quaking and trembler mice. There was little difference in the relative distributions of the 14K, 17K, 18.5K, and 21.5K forms of BP between control and md rat spinal cord homogenates at the ages examined. PLP content was reduced more than that of the other proteins in the md mutants, because it could not be detected by a technique capable of detecting 0.2% of the control brain level and 0.1% of control spinal cord level. This suggests that the expression of PLP may be preferentially affected in the md mutation.     

Characterization of Tubulin in Mouse Brain Myelin

Analysis of mouse brain myelin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that in the high-molecular-weight range it contained, besides the Wolfgram protein doublet, proteins comigrating with actin and with both subunits of tubulin. The occurrence of these alpha and beta subunits was confirmed by peptide mapping in myelin analyzed by two-dimensional electrophoresis. This tubulin did not arise from an artifactual binding of soluble brain tubulin to the myelin fraction: addition of exogenously labeled tubulin to brain homogenates proved that during myelin isolation by the procedure of Norton and Poduslo (1973) the contaminating tubulin was washed out. On the other hand, the distribution of tubulin isoforms in myelin was investigated by isoelectric focusing and compared with the distribution of the 21 isoforms listed for the whole brain soluble tubulin. It was shown that many isoforms were found in myelin (three isoforms for the alpha subunit and nine for the beta subunit), and that some isoforms were represented both in myelin and in soluble tubulin, but in different relative proportions.     

Retinoic Acid-Induced Differentiation of Human Neuroblastoma SH-SY5Y Cells Is Associated with Changes in the Abundance of G Proteins

Abstract: Western blot analysis, using subtype-specific anti-G protein antibodies, revealed the presence of the following G protein subunits in human neuroblastoma SH- SY5Y cells: Gaα, G_iα1, G_jα2, G_cα, G_zα, and Gβ. Differentiation of the cells by all-trans-retinoic acid (RA) treatment (10 μmol/L; 6 days) caused substantial alterations in the abundance of distinct G protein subunits. Concomitant with an enhanced expression of μ-opioid binding sites, the levels of the inhibitory G proteins G_iα1 and G_jα1 were found to be significantly increased. This coordinate up-reg- ulation is accompanied by functional changes in μ-opioid receptor-stimulated Iow-K_m GTPase, μ-receptor-mediated adenylate cyclase inhibition, and receptor-independent guanosine 5′-(βγ-imido)triphosphate [Gpp(NH)p; 10 nmol/ L]-mediated attenuation of adenylate cyclase activity. In contrast, increased levels of inhibitory G proteins had no effect on muscarinic cholinergic receptor-mediated adenylate cyclase inhibition. With respect to stimulatory receptor systems, a reciprocal regulation was observed for prosta- glandin E₁ (PGE₁) receptors and G_sα, the G protein subunit activating adenylate cyclase. RA treatment of SH-SY5Y cells increases both the number of PGE₁ binding sites and PGE₁ stimulated adenylate cyclase activity, but significantly reduced amounts of G_zα were found. This down- regulation is paralleled by a decrease in the stimulatory activity of G_zα as assessed in S49 cyc- reconstitution assays. However, the reduction in G_aα levels had no effect on both intrinsic and receptor-independent-activated [Gpp(NH)p or forskolin; 100 μtmol/L each] adenylate cyclase, suggesting that the amount of G_zα is in excess over the functional capacity of adenylate cyclase in SH-SY5Y cell membranes. Additional quantitative changes were found for G_zα, G_cα, and Gβ subunits. In contrast, neuronal differentiation in the presence of 12-O-tetradecanoylphor- bol 13-acetate (16 nmol/L; 6 days) failed to affect G protein abundance. Our results provide evidence for a specific RA effect on the abundance of distinct G protein sub- units in human SH-SY5Y neuroblastoma cells. These alterations might contribute to functional changes in transmembrane signaling pathways associated with RA-in- duced neuronal differentiation of the cells.     

Functional Coupling of G Proteins to Endothelin Receptors Is Ligand and Receptor Subtype Specific

1. The aims of the present study were (a) to determine the identity of the G proteins with which the endothelin receptor interacts and whether this interaction is subtype specific and (b) to determine whether agonist exposure can result in specific coupling between the endothelin receptor and G proteins.2. Coupling between endothelin A (ET_A) or endothelin B (ET_B) receptors and G proteins was assessed in two fibroblast cell lines, each expressing one receptor subtype. Four ligands, ET-1, ET-3, SRTXb, and SRTXc, were used for receptor stimulation. The G protein -subunit coupled to the receptor was identified by immunoprecipitation with an antibody against the endothelin receptor and immunoblotting with specific antibodies against different G protein -subunits.3. Unstimulated ET_A and ET_B receptors (ET_AR and ET_BR, respectively) were barely coupled to G_o. The unstimulated ET_AR coimmunoprecipitated with G_i3, whereas the unstimulated ET_BR was much less strongly coupled to G_i3. The coupling of ET_BR to G_i1G_i2 -subunits was much stronger than the coupling of ET_AR to these -subunits. Stimulation with the different ET agonists also resulted in differential coupling of G proteins to the receptor subtypes. All four ligands caused a strong increase in coupling of the ET_BR to G_i3, whereas coupling of the ET_AR to this subunit was not affected by ET-1 and was even decreased by SRTXc. On the other hand, all four ligands caused a much greater increase in the coupling of ET_AR to G_q/G₁₁ than in the coupling of ET_BR to these -subunits. Ligand-induced coupling between the receptors and the G_i1 and G_i2 -subunits is similar for the two receptor subtypes. The same was true for ligand-induced coupling of the receptors to G_o, except that ET-3 increased the coupling of this -subunit to ET_BR and decreased the coupling to ET_AR. Taken together, the results of this study show that coupling between ET receptors and G proteins is ligand and receptor subtype specific.4. It remains to be established whether this diversity of receptor–G protein coupling is of relevance for the various endothelin signaling pathways and/or pathological states.     

Acylation of Rat Brain Myelin Proteolipid Protein with Different Fatty Acids

The acylation of rat brain proteolipid protein (PLP) with tritiated palmitic, oleic, and myristic acids was studied in vivo and in vitro and compared with the acylation of lipids. Twenty-four hours after intracranial injection of [3H]myristic acid, only 16% of the PLP-bound label appeared as myristic acid, with 66% as palmitic, 9% as stearic, and 6% as oleic acid, whereas greater than 63% of the label in total or myelin phospholipid was in the form of myristic acid. In contrast, after labelling with [3H]palmitic or oleic acids, 75% and 86%, respectively, of the radioactivity in PLP remained in the original form. When brain tissue slices were incubated for short periods of time, the incorporation of palmitic and oleic acids into PLP exceeded that of myristic acid by a factor of 8. In both systems and with all precursors studied, the label associated with PLP was shown to be in ester linkage. The results suggest a preferential acylation of PLP with palmitic and oleic acids as compared with myristic acid. This is consistent with the fatty acid composition of the isolated PLP.     

Over-Expression of the DM-20 Myelin Proteolipid Causes Central Nervous System Demyelination in Transgenic Mice

Abstract: We have created transgenic mice bearing varying copy numbers of a transgene coding for normal DM-20, the alternatively spliced quantitatively minor isoform of myelin proteolipid protein. Demyelination of the CNS occurs as a consequence of 70 copies of this transgene. Overt symptoms begin at ∼3 months with a wobbling gait. Occasional seizures lasting a few seconds begin at 3–4 months. These symptoms progress in severity with age. Death occurs by 8–10 months. Myelination in 2-month-old animals, before the onset of any overt symptoms, appears morphologically normal at the electron microscopic level. However, the myelin in these 2-month-old animals has a reduced amount of the major myelin proteolipid protein and about three times as much DM-20 as normal animals. In 7-month-old animals that appear to be undergoing demyelination in the CNS, both the major myelin proteolipid protein and DM-20 are greatly reduced relative to the 2-month-old animal. Mice with 17 copies of the transgene also have a reduced amount of the major myelin proteolipid protein but appear to be otherwise normal and have normal life spans (>2 yr). Mice with low copy numbers of the transgene (2–4 copies) appear to be unaffected and have normal life spans.