An inborn error of cerebroside biosynthesis as the molecular defect of the jimpy mouse brain

Vol. 8, Part II, pp .911-918, 1989 . e~S~c ~nc~s n rent Britain

Pergamon Press

AN INBORN ERROR OF CBRSBROSIDS BIOSTNTHBSIS AS THR MOLECULAR DBFBCT OF T~ JILT MOUSS BRAIN Claudio 6a111, harry M. Kneebone' and Rodollo Paoletti Instituts of Pharasaology, University o! Milan, Italy sad Institute oP Pbaraaoology, University o! Cagliari, Ytaly (Received 17 May 1989) Bioaha~iaal investigations o! the Central Nervous Systea (CNS) in genetically deteraiued disorders, priaarily involving brain setabolisa, provides a sans o! elucidating noraal biocheaical proaeases . In this regard the "J~y" stock o! nine, a sez-linked single gene

sutaat in which lack o! syslia deposition has been reported as the basis of the neurological defeat (1), serves as a aodel for such a study . In previous investisations lroa this (2) and other laboratories (3) a aarked decrease in galaatolipids was observed together with essentially norsal values for the other lipids . The purpose o! the psbaent investigation ras to establish whether the defeat results asialy lros dea~ysliaatioa as proposed by Sidsan st al . (4) or iron inpsiraent of myelin loraation, as subsequently suggested (2) .

vii incorporation o! radioactive l~C-1 galaatose

The "in

into brain lipids was lolicved with particular referanae to labeling o! asrebrosides and sulphatides . Methods Five "Jiapy" nice, 18-20 days old, with evidence of the neurological disorder and ~ littersate controls were injected iatraperitoneally (5) tirith ~C-1 galaatose, 250 ua/kg. The anisais xare + Present address : Dept . of Child Health, Adelaide Children's Hospital, Adelaide, Australia

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sacriliaed 8 hours after the injectioa, blood samples rare taken to ezaluds diflereaaes is blood uptake of radioactivity and lipid eztraatsd frame individual braise (6) . Aliquots of brain lipid aztraats tars analysed using thin-layer ahra~atography (03) sad the sbseaae of cerebrosides in lipids Eros the "JiaPy~ aiae sass canataatly aanfirmed . The lipid sztracts of the tro groups xere then enah pooled !or lumber quantitative analysis . Preliminary ah:~ostatography oa Sephadez 6-25 columns ras performed to remove aoa-lipid labelled can+~~+marts (7) . The purified lipids rare fraatioaated by DBAS-cellulose aolusa ohroaatography and, aubsequeatly, the fractions obtained Eros the aoluaa, rare further analysed using thin-layer chromatography (8) . The individual]ipid classes thus separated xere qusatitated by the method o! phospho:vs deterainatiaa There appropriate (9) " Sterols, asrsbrosides, free tatty acids and aenaide rare determined using the method o! quantitative charring (10), sad sulphatides by a speatrophotoastria method (il), in each case folloriag this-layer ohroaatographic purilioatioas. The radioactivity o! the various lipid classes ras measured after thin-layer ch:waatographio separation and collsctio~n o! the individual bands o! the lipid aoataining adsorbent into counting vials. One milliliter of benzene-aethaaol (1 :1) ras added to the ponder and 10 al . o! a liquid scintillation mutate (65 g . POP, 130 ~ CH 3-POPOP, 104 g. naphthalene, 500 ml diozane and 500 al toluene) . This method, is our hands, proved to be better than alternative methods based a~n elution or eztraatioa of the compounds frost the adsorbent end gave a counting efliaieacy of 70~. The distribution of radioactivity betresa the hezose sad lipid portion of the aerebrosids molecule ra: deteraiaed sfter chromatogrnphia separation of the compound, acid hydrolysis and measurement of the ratio betreea radioactivity in the aqueous phase and in the

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91E

orgaaia phase . Results sad disaussioa The radioactivity recovered is the purilled lipids, is respoot of the total radioactivity e:traated tree brain rich ohlo~rotorsiaathaaol adstures, sad of the ia~oated radioactivity, and the speoi tia activity of tho pun lipids are si~aa is table l. A sodest ditferenoe aaly in the specitia activity o! the puritiod lipids appears troy the table. TABLE l . Total Radioactivity of Puritied Braies Lipids . Peraentage ot the C/M eztraated activity

Peraentage ot ia~eoted activity

S~scitia aativity (DPlI/ag)

Control

75 .2

0.178

687

Jiapy

75 .7

0.182

560

C/M . ahlorolara/sethsaol The bs+aia lipid distribution is the various classes sad their spsailia activities are presented is table 2, rhiah shore a virtual absaaae of aerebrosides sad a aiarlcsdly reduoed sulphatide aaatent is "J~y" mice awipared rich aoatrols . Ia all other lipid classes the aonceatration oP individual corpouadsis siailar in the tro groups . The total iaoorparatiaa o! Cik labelled galactosa ras essentially ideatiaal is each Broup mile distribution o! the ia-

aorporatian o! Cl~ labelled gaiaataso into individual brain lipids ditlersd anrkedly . Inaorporatian o! radioactivity actor iajeatia~ of Lbelied galactose ooourred is aü the lipid classes analysed, iadiaatigg

that galactose "ia vivo" cas provide the preatirsors for the los~aa tion of the various oanpo~neata o! these oaagtla: lipids . Tha highest spe~aitio nativity is lonad in the aarebrasides is the

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Vol, 8, No .18

acntrol group, thus indiaatiag its specific irraorporatian as " a galaatose unit" into galaotolipids . In aoatrast to the high incos~ poratioa of radioactivity in the aereba~osides of the control group "trace" amounts o! asrebroaide o! the "Jiapy" group precluded gnantitative uasurement cad thus prevented aalaulatioa o! the specific activity free the aouats detected (table 2) . TABLE 2 . Lipid Classes as Percentage of Brain Total Lipids cad Specific Aativitüs is Control (C) cad Jimpy (J) Groups

Psraeatags o! total lipids

Specific activity (DPIi/Ag lipid)

C

J

C

J

Cerebroaide

6.4

trace

1070

-

Sulphatida

1.4

0.6

251

137

Sterol

20 .8

19 .0

189

61

Phosphatic~yletàaaoLmiae

22 .6

25 .3

595

557

Phosphatidylaholiae

29 .5

29 .4

653

752

Sphiagapelin

1. 7

1. 3

224

39 2

Lysoleaithia

trace

0.9

-

-

Phosphatidglinositol, oardiolipia, phoephatidia acid cad aeramide vex also aeasured and found to be in the sane conaentrntions is both groups cad their specific activity insignitioaat. Phosphatidylserine is cot reported siaae it ws Pound to be the same is both groups in an earlier investigation (2) . The e:tsat of the ditterence is total incorporation o! radioactivity into aerebrosides betuean the two groups is shown in table 3 . Signitiaaat di!lerenaes in specific activity are obseswed in several lipid classes wàea the txo groups are aoapared. In the "Jimpy" group the specific activities of sulphatides and sterol are lower and that of sphingoayelia is higher than the values of the

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CEREHR06mE HI06YNTHEBIS

915

TABLB 3 . Total Incorporation of Radioactivity into Cerebrosides sad Sulphatides is the Control (C) and Jirpy (J) stoups

DPK/gras o! braie lipids

Control Jirpy

Cerebroside

Ratio C/J

68,100 ~,,83o

14 :1

Sulphatide 3,560 szz

Ratio C~J

~r .3 :1

aorreapoading lipid classes in the aantrol group. Incorporation of isotope into giycerolphospholipids, phosphatidyiethanolarins and phosphatidylcholine instead is either is each group. It is o! aonaid,erable interest to note that although csrebrosides are virtually absent in the "Jiapy" rise sulphatide is present is detsatabie arounts . Althoagh this finding dearads further iavestigatioa it is possible to postulate a hypothesis fror the observed data . Sulphatides are forted is riorosomes lroe cersbroaides xhiah are predoriaaatly utilized for agrelin lorratioa . Sulphatides are aUo present is aprelin xith a cerebroside: sulphatide ratio of 5:1 (lz) . However they have been found in high aonaentrstion in eon-~relia preparations (13) . It is thus likely that the stall arount o! aerebroaides forted in the "Jirpy" rice is insufficient to provide the correct proportion !ar norral fasmtioa o! gelia. It is therefore freely ooavsrted to sulpbatide, thereby producing the relatively

high apecitic nativity of the rsdvaed quantity toned. This interpretation is supported by the data reported in table 2. The asaoaiatloa bstweea the defect in galactolipid foraatloa and the observed reduction in the apeaitia activity o! the cholesterol fraction aanforrs xith the current viw o! gelin forration. It has been ahonn that although gelin aaatained aü the saran brain lipids, cholesterol, aersbrosides and phosphatidyl-

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ethanolamine predominated (12) . It has been proposed that the myelin lipid sub-cult is cooposed of sphingolipid-aholeaterol and phospholipid-cholesterol complezes (14), while soae of the non-cholesterol lipids may remain uacomplezed (13) .

This concept suggests that a

correlation exists betxsen the rats o! cholesterol and galactolipid formatiaz, as indicated by the results obtained xith the " Jimpy " mice . The incorporation of radioactivity into sphingomyelin is higher in the "Jimpy" group thta in the controls . Since sphingowyelin and cerebrosides both derive Eros the common precursor sphingosiae, the block is cerebroside formation could enhance the conversion o! sphingosine to aphingoayelin and explain the higher specilic activity found in the " JimPy" group. To exclude the possibility that the differences in 14 C uptake by the cerebroside !motions of the txo groups could be esplained by an alteration in the form of incorporation of 14C, the distribu tia~n of radioactivity betxeea the hezose and lipid portion oP the cerebroside molecule has been evaluated as described in the methods . If the 14C-hezose xere metabolized and incorporated in a random manner the anticipated ratio C6 hezose fragment/C42 ceramide fragment *ould be less than, or equal to, 0 .14 (5) . A ratio larger than 0.14 indicates preferential incorporation of the labelled carbon into the hezose moiety . This ratio was found to be 0 .49 for the controls and 0 .2~ for the "JisPy" grasp . The results indicate therefore that a preferential incorporation of 14C hezose in cersbrosides exists for both groups . The greatly decreased biosynthesis oP cerebroaides in the " Jigpy" nice and the utilisation of galactose for other metabolic processes may reduce the availability of Lbelled galactose for direst incorporation into aerebrosides in the "Jimpy" group as indicated Dy the lacer ratio. From the data presented it is possible to locate the basic defect in the " Jimpy" brain . The conversion of sphingosine to cerebrasides is greatly reduced and ßyelin deposition is conaequeatly

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CEREBROSiDE BI06YNTHESIS

917

impaired . The changes observed in the biosynthesis of other lipid classes appear to be a consequence of the specific primary defect in aerebroside Formation. The data obtained in the "Jimpy" lice indicate that myelin foraatioa is an ezaat process requiring close metabolic correlation between its eonponents . Thus, in this regard the "Jimpy" mouse serves as a useful model for further studies of disorders of this aspect of brain lipid metabolism . Acknowledgement This research was supported in part by the United Cerebral Palsy Research and Education Foundation, Inc ., New York, U .S .A . and by the Association for the Aid to Crippled Children, New York, U.S .A . References R .L . SIDMAN, M.M . DICKIS, S .H . APPEL, Science , 1~, 309 (1964) 2.

C . GALLI, D. RS CSCCONI GALLI, Nature , _220, 165 (1968)

3.

J .L . NUSSBAUM, N .M . NSSKOVIC, D.M . KOST

P . MANDEL, Bull .

Soc. Chim . Biol . , 50, 2194 (1968) 4.

R.L . SIDMAN, R . HAYSS, J. Neuropath . Sxp. Neurol . , 24, 173 (1965)

5.

R.M . BURTON, M.A . SODD, R.O . BRADY, J. Biol . Chem . , _233, 1053 (195s)

6.

G. ROUSSR, G. KRITCHSVSKY, C . GALLI, A. YAMOMOTO, A.G . KNUDSON, Jr ., in " Inborn Disorders of Sphingolipid Metabolism" , p. 303, Pergamon Press, Ozford & New York (1966)

7. 8.

A.N . SIAKOTOS, G . ROUSSR, J . Am . Oil Chew . Soc. , 42, 913 (1965) G. ROUSfiR, G. KRITCHSVSKY, C . GALLI, R . HELLER, J . Am . Oil Chem . Soc . , 42, 215 (1965)

9.

G. ROUSSR, A N. SIAKOTOS, S . FLSISCHSR, Lipids , l, 85 (1966)

10 .

J .B . MARSH, D .B . WEINSTEIN, J . Lipid Res . , 7, 574 (1966)

11 .

S .L . KSAN, J. Lipid Res. , 9, 319 (1968)

12 .

W.T . NORTON, L .A . AUTILIO, J . Neurochem. , _13, 213 (1956)

13 .

A .N . DAVISON, N.A . GRSGSON, 9lochem. J . , 85, 558 (1962)

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CEREHR08TDE HIQBYNTHESI3

14.

F .A. 1IMDSNHBWBL, J .A.O .C.S. , 40, 455 (1963)

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L.F, S11C~ B.P. èfOBLS, Lipids , 3, 157 (1968)

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