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Experimental Neurology 159, 258–266 (1999)Article ID exnr.1999.7132, available online at http://www.idealibrary.com on

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Biochemical Analysis of Myelin Lipids and Proteins in a Model of MethylDonor Pathway Deficit: Effect of S-Adenosylmethionine

Roberto Bianchi,* Federico Calzi,* Sara Savaresi,* Roberto Sciarretta-Birolo,* Raffaele Bellasio,*Virginia Tsankova,† and Maria Teresa Tacconi*

*Department of Molecular Biochemistry and Pharmacology, Istituto di Ricerche Farmacologiche Mario Negri, 20157 Milan, Italy;and †Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Sofia, Sofia, Bulgaria

Received December 4, 1998; accepted May 8, 1999

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S-Adenosylmethionine (SAMe) is the methyl donoro numerous acceptor molecules. We used cyclo-eucine (CL), which prevents the conversion of methio-ine to SAMe by inhibiting ATP–L-methionine–adenos-ltransferase (MAT), to characterize the lipid androtein changes induced in peripheral nerve and brainyelin in rats during development. We also investi-

ated the effect of exogenous SAMe by administeringAMe-1,4-butane disulfonate (SAMe-SD4). CL was givenn days 7, 8, 12, and 13 and SAMe-SD4 was given dailyrom day 7; the animals were killed on day 18. CLccumulates in the brain reaching a concentrationithin 24 h compatible with its ID50 in vitro and

nteracting with methionine metabolism; brain MATctivity and SAMe levels were lower and methionineevels higher than in controls. CL significantly reducedrain and nerve weight gains, brain myelin content,roteins, phospholipids, and galactolipids. Amonghospholipids in nerve and brain, only sphingomyelinas significantly increased, by 35–50%. Sciatic nerverotein analyses showed some significant changes:rotein zero in sciatic nerve remained unchangedut the 14.0- and 18.5-kDa isoforms of myelin basicrotein showed a dramatic increase. Among theain proteins, in purified brain myelin, the pro-

eolipid protein and dimer-20 isoform decreasedfter CL. SAMe-SD4 highlights some sensitive param-ters by counteracting, at least partially, somelterations of PL—particularly galactolipids andphingomyelins—and proteins induced by CL.he partial beneficial effects might also be ex-lained by the age-related limited bioavailability ofxogenous SAMe, a finding, to our knowledge,ot yet reported elsewhere. This study demon-trates that availability of methyl donors is closelyelated to the formation of myelin components. r 1999

cademic Press

Key Words: rat; brain; sciatic nerve; hypomyelin-tion; cycloleucine; myelin protein; myelin lipid.

M

258014-4886/99 $30.00opyright r 1999 by Academic Pressll rights of reproduction in any form reserved.

INTRODUCTION

Methyl group deficiency appears to be a pathogenicechanism in various genetic, neurological, and psycho-

ogical disorders, including dementia, depression, mul-iple sclerosis, AIDS–dementia complex, and subacuteombined degeneration of the spinal cord (SCDC; 5).any of these diseases involve a myelination defect

nd demyelination is found in most cases of inbornrrors of metabolism affecting the methyl transferathway, suggesting that methylation is important forhe integrity of the myelin sheath (22, 30, 43). Methyl-tion impairment may affect myelin lipid and proteinontent and composition. Phosphatidylcholine (PC) de-ives its choline partly from methyl transfer via phos-hatidylethanolamine N-methyltransferase (PEMT, EC.1.1.17; 4, 17, 56, 57) and various PEMT isoforms wereormed in rat brain myelin (58). Some myelin proteinsontain methyl groups which seem to affect theirunctions. Myelin basic proteins (MBP) contain a meth-lated arginine residue that increases the hydrophobic-ty of the protein, thus influencing how it interacts withhospholipid molecules in the formation of the myelinheath (6). Inhibitors of the methyl transfer enzymesroduce a less compact myelin in oligodendroglial cellsnd the degree of methylation correlates with anncrease in the interaction of MBP with lipid vesicles18, 31). The levels of MBP-specific methylase activityre temporally correlated with myelination duringrain development (11, 16, 27) and methylase activityas depressed in brain from dysmyelinating mutantice (32, 51). S-Adenosylmethionine (SAMe) is theethyl donor to numerous acceptor molecules likeNA, phospholipids (PL), and proteins. SAMe synthe-

is may be impaired: (i) if the supply of methionine iseduced, like during methyl tetrahydrofolate or vita-in B12 deficiency (1, 13, 30); and (ii) if the activity ofTP–L-methionine–adenosyltransferase (MAT; EC.5.1.6) is defective (42, 59). This enzyme is present inifferent isoforms: MAT I and III are found in liver, and

AT II is found in all other tissues, including brain (2,

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259MYELIN LIPIDS AND PROTEINS IN HYPOMETHYLATION

3). Subjects deficient in hepatic MAT activity haveeen described (22, 25, 26, 28), and a lesion in theirAT1 gene has been reported (59). People who com-

letely lack MAT I/III activity show neurological abnor-alities, suggesting that hepatic MAT may be impor-

ant for myelin development (10). Treatment withAMe improved the patient’s conditions in one case ofAT deficiency (54) and was efficacious in other brain

isorders possibly involving impairment of methyl do-or capacity (9). In this study we used 1-aminocyclopen-ane-1-carboxylic acid (cycloleucine, CL), an analog ofethionine which prevents its conversion to SAMe by

nhibiting MAT activity (38, 39), to characterize lipidnd protein changes induced in sciatic nerve and brainyelin in developing rats. Chickens injected with CL

eveloped cerebrum vacuolation of myelin similar toumans with vitamin B12 deficiency (53) and earlytudies found that CL produced CNS lesions in miceith a pathology similar to human SCDC (24). In adultice CL induces primary degeneration of axons in the

erebellum, rostral spinal cord, and peripheral nerves46). In 7-day-old mice treated with CL, axonal abnor-alities (sciatic and tibial nerves) were marked. Theice also showed morphological and electrophysiologi-

al alterations in intramuscular nerves and nerveerminals and a dramatic reduction in twitch andetanic responses, producing a picture of distal motorxonopathy (19). In addition, dorsal root ganglion cellsresented dense bodies and degenerated organellesfter 72 h (36). Confirming early data in mice (46),L-treated rats developed a state similar to that seen

n B12-deficient SCDC which includes changes in brainnd spinal cord PL content and myelin splitting (50).rom the biochemical point of view, CL reduces the

ncorporation of methyl groups into methylarginine inBP (53). On the basis of these observations we started

n integrated study of CL toxicity to the nervousystem in suckling rats. We also investigated therotective effect of SAMe-1,4-butane disulfonate (SAMe-D4) administered with CL.

MATERIALS AND METHODS

nimals

Procedures involving animal and their care are con-ucted in conformity with the institutional guidelineshat are in compliance with national (D.L. No. 116,.U., suppl. 40, 18 febbraio 1992, Circolare No. 8, G.U.,4 luglio 1994) and international laws and policiesEEC Council Directive 86/609, OJ L 358,1, Dec. 12,987; ‘‘Guide for the Care and Use of Laboratorynimals,’’U.S. National Research Council, 1996). Seven-ay-old rat pups (CD, Charles River Italy, Calco, Italy)ere caged with their mothers and exposed to a 12-hay–night cycle. Pups were divided into four groups:

roup 1 received 400 mg/kg of CL (Sigma, St. Louis, P

O), ip, on days 7, 8, 12, and 13 after birth; group 2eceived CL, as above, and 100 mg/kg SAMe-SD4BioResearch, Liscate, Italy), ip, daily from day 7 to day7; group 3 received SAMe-SD4, as above; and group 4eceived saline. For each experiment two to threeitters were assigned to each group; experiments wereepeated at least twice. At the preset times animalsere killed by decapitation under ether anesthesia.issues were quickly removed, weighed, and divided

nto aliquots. Brain and sciatic nerves were stored at80°C for subsequent analysis. Brain aliquots wereomogenized in 0.32 M sucrose to measure CL and-methionine and to prepare myelin. For SAMe determi-ation one brain aliquot was homogenized in 0.2 Merchloric acid. In some experiments animals wereilled at different times after CL to measure brain

evels of CL, L-methionine, and SAMe.

nalytical Methods

Proteins in tissue preparations were determined byhe procedure of Lowry et al. (40) with bovine serumlbumin as the standard. Brain homogenates in 0.32 Mucrose were centrifuged at 156,000g for 30 min torepare cytosol to measure the contents of CL and-methionine, using an amino acid analyzer (BeckmanA 6300). MAT activity was measured in the sameytosol preparation by a radioisotopic method (12).yelin was prepared by gradient centrifugation (47).ipids in myelin and sciatic nerves were extracted inhloroform:methanol (23). Samples of the chloroformhases, containing lipids, were used to determine Pi inL after acidic digestion (55) and galactolipids (62). PL

ractions were separated by TLC (silica gel 60, withoncentration zone, Merck), using chloroform:ethanol:riethylamine:water (30:33:35:8) as the developing mix-ure. Each fraction was identified by authentic stan-ards run in parallel; silica in spots corresponding toL fractions was scraped off and used for Pi determina-ion. Brain SAMe content was measured by HPLCethod (60). The proteins from nerve homogenates and

rain myelin (1:20 w/v; 10 mM Tris–HCl, pH 7.4,ontaining 1% sodium dodecyl sulfate, SDS) were sepa-ated using 12% SDS–polyacrylamide gel electrophore-is (SDS–PAGE) according to the method of Laemmli35) and gels were stained with Coomassie blue (21).or each lane 50 µg of total protein was applied. Touantify changes, the intensity of each protein band onhe gel lane was determined by densitometric scanningnd expressed as percentages of the total loaded on theane (Ultroscan XL, LKB, Bromma, Sweden) and calcu-ated as follows: (intensity of the desired band) 3 100/total intensity of the material in the lane). Bands withhe same apparent molecular weight in different lanes,n treated and untreated groups, were then compared.

hen indicated, separated proteins were transferred to

VDF membranes for immunoblotting analysis and

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260 BIANCHI ET AL.

ested with specific antibodies. Nonspecific proteininding sites were blocked for 1 h at room temperaturer overnight at 4°C with blocking buffer composed of 4%onfat dried milk in phosphate-buffered saline. Therimary antibodies were directed against 28,38-cyclicucleotide 38-phosphodiesterase (CNPase; Sternbergeronoclonal Incorporated, Baltimore, MD), MBP (Boeh-

inger Mannheim), protein zero (P0), and proteolipidrotein and dimer-20 (PLP; DM-20; kind gift from Dr.. H. Koeppen, Albany, NY). A chemiluminescent detec-

ion procedure was used (ECL Western blotting proto-ol, Amersham, Buckinghamshire, UK).

tatistical Analysis

Results are expressed as means 6 SE. The signifi-ance of any differences was evaluated by two-waynalysis of variance (ANOVA) followed by Dunnett’sest and multiple comparisons were made using Tukey’sighly significant difference test.

RESULTS

Our studies focused on the simultaneous changes inhe major myelin-associated lipids and proteins duringhe critical period of early development, up to 18 daysfter birth, when active myelination take place; weave not yet extended our investigation to the effects ofethylation damage over the life span. Body weight

ain was significantly reduced in CL-treated pups inomparison to controls (15.3 6 0.4 vs 23.5 6 0.5,, 0.05). In the group receiving SAMe-SD4 1 CL,

ody weight gain was greater than CL-treated animals,ut less than those given SAMe-SD4 alone (20.6 6 0.5s 26.7 6 0.4). Brain weights (n 5 6) were lower (25–0%) in CL and SAMe-SD4 1 CL-treated pups than inontrol and SAMe-SD4 rats.To monitor the uptake and level of CL in brain,

nimals treated with CL and saline on days 7, 8, 12,nd 13 after birth were killed at different times afterreatments (Fig. 1A). CL accumulated in brain cytosolnd its concentration remained elevated up to 18 daysfter birth (between 1.5 to 2.0 µmol/g brain); thisoncentration is comparable with the IC50 of CL (1.66M) obtained in vitro on brain MAT (data not shown).Methionine levels in the brain cytosol were elevated

n average by 40–50% throughout the experiments, inccordance with a possible inhibition of MAT activityFig. 1B). MAT activity, measured directly in brainytosol of saline and CL-treated pups, was reduced by6, 17, and 28% in CL-treated animals, in comparisono controls, respectively, 9, 14, and 18 days after birthTable 1).

The SAMe concentration in brains of CL-treated ratsas significantly lower than controls already on day 9,fter the first set of CL injections, and remained low

hroughout the experiment. Table 2 shows the effect of

xogenous SAMe. Two hours after SAMe-SD4 injection,AMe rose significantly in control and CL-treatedrains of 9- and 14-day-old pups, demonstrating thathis compound passes the blood/brain barrier. Whenats reached 18 days, SAMe-SD4 no longer raised brainAMe.The amount of myelin recovered from brain of CL-

reated rats was 45% less (Table 3). Analyses of myelinocusing on protein and polar lipids (PL and galacto-ipid) showed significant reductions in CL-treated ratsf 55.4, 34.7, and 50%. SAMe-SD4 raised galactolipidontent both in control and in CL-treated rats by 40%ut had no effect on protein and PL.

FIG. 1. Brain concentrations of cycloleucine (A) and methionineB) in brain of suckling rats receiving saline or cycloleucine (500g/kg intraperitoneally) on days 7, 8, 12, and 13 after birth.

TABLE 1

Methionine Adenosyltransferase Activity of Cytosol fromrain of 9-, 14-, and 18-Day-Old Rats Receiving Saline (C) orycloleucine (CL; 500 mg/kg Intraperitoneally), Once a Dayn Days 7, 8, 12, and 13 after Birth

Agedays)

Brain cytosol methionineadenosyltransferase activity

(nmol/mg protein/30 min 6 SEM)

C CL

9 4.90 6 0.02 3.62 6 0.03*14 4.55 6 0.02 3.81 6 0.01*18 5.33 6 0.25 3.82 6 0.08*

Note. n 5 6; * P , 0.05 vs respective control.

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261MYELIN LIPIDS AND PROTEINS IN HYPOMETHYLATION

Weights of sciatic nerves were significantly lowerhan controls after CL, with a consequent reduction ofotal proteins and PL, but when their concentrationas expressed as milligrams per gram of nerve the

eduction was no longer evident. SAMe-SD4 supplemen-ation to CL-treated rats only partially reduced the lossf weight and protein content (data not shown). PLomposition of brain myelin and sciatic nerves showedimilar patterns in both tissues. The distribution of PLractions was not affected by CL, except for sphingomy-lin, which was significantly higher (50%) than controldata not shown). Sciatic nerves presented a 35%ncrease in this lipid. SAMe-SD4 tended to reduce thencreases but did not restore control values.

About 30 protein bands (Fig. 2) were observed in theel from sciatic nerve homogenates after electrophore-is and Coomassie blue staining; as expected, theseonsisted of a large amount of P0 followed by a 66-kDarotein and MBP (different isoforms ranging from 21.5o 14.0 kDa). At least two main differences are alreadylear from the gel, namely, the smaller 66-kDa proteinand in the CL-treated group than the control one and

TABLE 2

S-Adenosylmethionine (SAMe) Concentration in Rat Brainf 9-, 14-, and 18-Day-Old Rats Receiving Saline (C), Cycloleu-ine (CL), S-Adenosyl-L-methionine 1,4-Butane DisulfonateSAMe-SD4) or CL 1 SAMe-SD4

Agedays)

Brain S-adenosylmethionine(nmol/g brain 6 SEM)

C CL CL 1 SAMe-SD4 SAMe-SD4

9 51.1 6 1.8 38.6 6 1.9*,** 50.7 6 1.5*,† 64.0 6 1.7*,†14 49.9 6 1.0 36.7 6 1.0*,** 44.7 6 1.7*,† 61.8 6 2.2*,†18 47.9 6 1.0 34.2 6 1.8*,*** 41.5 6 3.1†† 49.6 6 0.8***,†

Note. Schedule of treatment: CL, 400 mg/kg intraperitoneally, onceday on days 7, 8, 12, and 13 after birth; SAMe-SD4, 100 mg/kg

ntraperitoneally, every day from day 7 to day 18; animals were killedh after the last SAMe-SD4 treatment. n 5 6–10. * P , 0.01 vs C;

* P , 0.01 vs SAMe-SD4; *** P , 0.05 vs SAMe-SD4; † P , 0.01 vsL; †† P , 0.05 vs CL.

TAB

Protein and Phospholipid Concentrations in Brain MyelinS-Adenosyl-L-methionine 1,4-butane Dis

C

ry weight (mg/brain)a 4.04roteins (mg/brain) 1.58 6 0.09 0.72hospholipids (mg/brain) 1.55 6 0.01 1.02alactolipids (µg/brain) 112 6 11 56

Note. Schedule of treatment: CL, 400 mg/kg intraperitoneally, onntraperitoneally, every day from day 7 to day 18; animals were killed.01 vs C; *** P , 0.05 vs SAMe-SD4; † P , 0.01 vs SAMe-SD4; †† P ,

a

Myelin dry weight was obtained from two pools of three brains.

he increase of MBP isoforms in CL-treated group,articularly of the 14 kDa; in both cases SAMe-SD4xerted some protection (Fig. 2A). These changes areore evident in Fig. 2B which compares a laser densito-etric tracing of one control and one CL lane (see

rrows at 66-, 14.0-, 17.2-, 18.5-, and 21.5-kDa bands).Quantitative data for some proteins (see Materials

nd Methods), accounting for about 70% of all materi-ls of the lanes, are given in Table 4. Although theistribution of P0 was unaltered for control (C), CL, andL 1 SAMe-SD4 (28.5, 27.9, and 27.9, respectively),everal changes were seen for other proteins. Some ofhem showed reductions in the CL-treated group, includ-ng the 116-, 90-, 66-, 58-, and 56-kDa bands. Otherolypeptides increased, namely, the 200-, 21.5-, 18.5-,7.2-, and 14.0-kDa bands. Among these the MBP withelative molecular mass of 14.0, 17.2, 18.5, and 21.5Da showed a dramatic increase, especially in the 14.0-nd 18.5-kDa isoforms (Figs. 2A and 2B).Besides identification by their relative molecularass, some proteins, including P0, MBP, and CNPase,ere also analyzed by immunoblotting employing spe-

ific antibodies. An example is given in Fig. 2B (inset) inhich quantitative data for the 14.0-kDa MBP bandre presented after electrophoresis, immunoblot, andensitometric measurements. These data are in agree-ent with those in Table 4. As shown in Table 5, the

roportions of the four MBP isoforms, relative to the1.5-kDa isoform, which was taken as equal to 1,iffered significantly in the CL-treated group fromontrols, the 18.5- and 14.0-kDa isoforms being 2.5 and.1 times higher. SAMe-SD4 restored the normal 18.5-nd 14.0-kDa isoform distribution without affecting theistribution of the 17.2-kDa isoform. Significant pro-ein alterations (more than 30 bands detected) werelso seen in brain myelin (data not shown). The quanti-atively predominant protein was at Mr 56–57 kDa, 46Da, a double band often referred to as W1 and W2ccording to Wolfgram (61), 30 and 20 kDa (PLP andM-20), and the two MBP bands at 21.5 and 18.5 kDa.

n the CL group the bands at 210 and 46 kDa (CNPase)

3

18-Day-Old Rats Receiving Saline (C), Cycloleucine (CL),fonate (SAMe-SD4) or CL 1 SAMe-SD4

CL CL 1 SAMe-SD4 SAMe-SD4

2.26 2.31 3.660.10**,*** 0.79 6 0.06**,*** 1.48 6 0.100.07*,*** 0.96 6 0.05*,*** 1.38 6 0.045** 78 6 9*,†,†† 157 6 9*

a day on days 7, 8, 12, and 13 after birth; SAMe-SD4, 100 mg/kgafter the last SAMe-SD4 treatment. n 5 6–8. * P , 0.05 vs C; ** P ,

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nd PLP and DM-20 were reduced. Other proteinsncluding 220-, 66-, 56-, and 41-kDa bands slightlyncreased, while MBP remained unchanged.

DISCUSSION

This study found that CL administered to sucklingats enters the brain, reaching 1.5–2.0 µmole/g brain,hese levels remaining constant throughout the experi-ent, compatibly with the inhibitory effect on MAT

ctivity. These changes lead to a reduction of MATctivity and SAMe in brain and to an increase in theontent of the substrate methionine, making it a reli-ble model of methyl donor deficiency, thus confirmingata from Lombardini and Talalay (39) showing thatL inhibited MAT activity in homogenates of several

issues, including brain. This altered metabolism pro-uces motor disturbances such as tremors and uncoor-

FIG. 2. Coomassie blue-stained SDS–polyacrylamide gel, densitomnalysis of the 14-kDa MBP from sciatic nerves. (A) 50 µg of proteinsycloleucine; lane 3, cycloleucine 1 SAMe-SD4; lane 4, molecular weights expressed in kDa). (B) Representative densitometric tracing

ats. Arrows indicate the main changes (see Results). The inset hisensitometric quantification in C (n 5 8), CL-treated (n 5 10), and CL

inated movements, impaired growth (indicated by s

ower body, brain, and sciatic nerve weight), and alter-tion of specific myelin lipids and proteins. Among thearious parameters we studied, myelin depositioneemed very sensitive to CL. The total recovery fromL-treated rats was only 45% of control, and its amountf lipids and proteins was even less. These resultsuggest that the availability of methyl donors is verymportant for myelin deposition.

It is not yet clear why the nervous system (andyelin sheath) is so sensitive to the deficiency ofethyl donors, which are required for an extraordinary

ariety of biochemical and physiological processeshroughout the body. The literature suggests that thextent of PC methylation may be important in myelinormation since PL polar groups interact with MBP,nd this may affect the membrane structure and func-ion (52). Under our experimental conditions the markededuction in the absolute PC content in myelin was

ric tracing of two lanes, and quantitative data following Western blots electrophoresed on 12% polyacrylamide gel: lane 1, control; lane 2,ght markers (side numbers are the respective apparent molecularSDS–PAGE separation of control (C) and cycloleucine (CL)-treatedram shows the 14-kDa MBP levels after immunoblot analysis andSAMe-SD4-treated (n 5 6) rats.

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263MYELIN LIPIDS AND PROTEINS IN HYPOMETHYLATION

ecessarily indicate that the PEMT pathway is irrel-vant in the nervous system, but rather that its deficitffects PL composition in a complex manner. AlthoughEMT activity is very low in brain (17, 57) compared,

or example, to the liver (37), PC intermediates—onomethyl-PE and dimethyl-PE—have been found in

at brain (56), and rat axons contain several PEMTsoforms (17, 58). A possible mechanism by whichEMT may affect brain PC synthesis, as suggested byagret et al. (41), is the important role of PEMT and

hospholipase A2, in cluster, in the transport of polyun-aturated fatty acids (PUFA)—which are not synthe-ized in the developing brain (19)—from the cerebralndothelium to astrocytes. This may explain the impres-ive reduction of PUFA-enriched PL in brain and sciaticerve myelin of CL-treated rats, while the relativeontent of sphingomyelin (poor in PUFA) is increased.ndeed, Ramsey and Fischer (50) found lower levels of

TABLE 4

Relative Proportions of Control (C), Cycloleucine (CL), andycloleucine 1 S-Adenosyl-L-methionine 1,4-Butane Disulfo-ate (CL 1 SAMe-SD4) Proteins of 18-Day-Old Rat Sciaticerves

ProteinsW (kDa)

C(n 5 8)

CL(n 5 13)

CL 1 SAMe-SD4(n 5 4)

00 1.5 6 0.1 2.0 6 0.3 1.6 6 0.216 0.9 6 0.2 0.6 6 0.1 1.4 6 0.10 1.4 6 0.1 0.7 6 0.1 0.9 6 0.16 12.7 6 0.8 8.1 6 0.8 9.2 6 0.48 6.8 6 0.3 4.1 6 0.4 6.7 6 0.26 1.5 6 0.1 1.1 6 0.2 1.2 6 0.18 P38 2.4 6 0.2 2.1 6 0.2 2.2 6 0.11 P0 28.5 6 0.9 27.9 6 0.9 27.9 6 0.41.5 MBP 2.1 6 0.1 2.4 6 0.1 3.0 6 0.18.5 MBP 4.3 6 0.2 5.9 6 0.2 5.5 6 0.37.2 MBP 3.8 6 0.1 4.4 6 0.2 5.0 6 0.14 MBP 4.0 6 0.2 7.0 6 0.8 5.9 6 0.4

Note. Samples were run at least in duplicate. For calculation of theercentages of each protein in total homogenates, refer to Materialnd Methods. Schedule of treatment, see legend to Table 3.

TABLE 5

Sciatic Nerve Myelin Basic Protein Ratio in Control (C),ycloleucine (CL), and Cycloleucine 1 S-Adenosyl-L-methio-ine 1,4-Butane Disulfonate (CL 1 SAMe-SD4) Proteins of8-Day-Old Rat Sciatic Nerves

Isoforms(MW, kDa): 21.5 18.5 17.2 14.0

1 2.0 1.8 1.9L 1 2.5 1.8 2.9L 1 SAMe-SD4 1 1.8 1.7 2.0

s

nsaturated FA in PC and PE in spinal cords ofL-treated rats aged 18 days than in untreated con-

rols, supporting this hypothesis.Recently galactolipids have joined the list of crucial

omponent of the myelin sheath (7 as review). Theirostulated functions include facilitative interactionetween myelin and axons and participation in myelinrotein traffic; they actively cooperate in membranetabilization (7). In addition, brain from galactolipid-eficient mice shows hypomyelination, regional myelinnstability, vacuolar degeneration, and myelin splitting8). These observations are interesting in view of theact that in CL-treated rats we observed a significantecrease of galactolipids, which was reversed by SAMe-D4 treatment. Intriguingly, SAMe-SD4 alone in-reases galactolipids in control rats.Data have been published showing that MBP, PLP,

nd P0 make an important contribution to the forma-ion of the myelin sheath (29). In CNS, where the MBPontent seems unaltered, we observed the largesthanges for PLP and DM-20, whereas in peripheralerve MBP were the most susceptible to CL treatment,ith a relatively stable P0 content. In rodents, severalBP isoforms are reported to be formed by alternativeNA splicing from a common primary transcript (14)nd four different molecular species with apparentolecular mass of 14.0, 17.2, 18.5, and 21.5 kDa have

een described in rats. They are thought to be impor-ant for maintaining the compactness of myelin in PNSnd CNS (14, 34). MBP isoforms are developmentallyegulated and the mouse brain showed low levels of the7.2 and 21.5 kDa and increased levels of the 14.0- and8.5-kDa isoforms with myelin maturation (3). Duringarly myelination (8–15 days postnatally) plasma mem-rane is formed and starts to wrap the nerve axonoosely; in a second stage (15–30 days) protein deposi-ion, maturation, and compaction take place (15). If thisecond process occurs earlier in development it may beeleterious (48). Our finding of a dramatic increase inhe 18.5- and 14.0-kDa isoforms in PNS is consistentith abnormal early expression of these MBP isoformsuring myelination, possibly leading to incorrect my-lin formation or maturation. The biological signifi-ance of protein methylation, a posttranslational eventhich occurs in a variety of proteins, has not yet been

learly explained (49). Data on methylation of myelinroteins are very scanty and mostly regard changes ofBP methylation. There are data showing that MBPethylation, by increasing their hydrophobicity, en-

ances interactions with lipid vesicles (6, 18, 31),nfluencing myelin compactness. The present studyndicates that SAMe-SD4 at least partially counter-cted some alterations of PL and proteins induced byL and highlights some sensitive parameters, probably

he most susceptible to change, during demyelination

tates suspected or known to be linked to methylation Note. Schedule of treatment, see legend to Table 3.

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eficiency. The partial beneficial effects might also bexplained by the limited bioavailability of exogenousAMe. Brain levels were increased 2 h after SAMe-SD4

n 9- and 14-day-old rats but not in 18-day-old ones. Tour knowledge, this is the first report of age-relatedAMe transport to the brain, the difference beingossibly due to changes in blood/brain barrier perme-bility during development. The synthesis of myelinomponents is under a tightly controlled program,ipids and proteins are highly integrated, forming aery stable ultrastructure, and a deficit of one compo-ent may lead to the coordinated downregulation of allhe others (44, 45). Hence myelin structure and func-ion are closely related to the contributions of thendividual lipid or protein components. In our experi-

ental model we found a selective susceptibility toither CL or SAMe-SD4 of some myelin components,ncluding MBP, PLP, and galactolipids. It will be inter-sting to see whether messenger RNAs of specificyelin component genes are affected or more efficiently

ranslated after methyl donor deficit and treatmentith exogenous SAMe.Another aspect worth investigat-

ng, considering the different MAT forms in differentissues, is whether CL acts on distinct enzymaticctivities affecting both protein and lipid methylation2, 10, 33, 58). The model we have started characteriz-ng will help us understand not only the mechanism(s)f the inborn deficit of MAT, whose nervous systemesions seem to become evident later in life (10), butlso more generally the pathological consequences ofther inborn and metabolic deficiencies of precursorsnd cofactors regulating methyl donor availability.SAMe methylases exert a variety of functions. Clari-

ying the importance of methyl donor availability forhe different myelin components thus holds promise forotential pharmacological approaches to methyl defi-its that lead to devastating abnormal myelination oremyelination.

ACKNOWLEDGMENTS

We thank Dr. J. Eichberg for critical reading of the manuscript anduggestions. The financial support of Telethon-Italy (Grant D 51) isratefully acknowledged.

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