Developmental changes of three phosphoinositide-specific phospholipase C isozymes in the rat nervous system

Developmental Bram Research, 59 (1991) 7-16 Elsewer

7

BRESD 51226

Developmental changes of three phosphoinositide-specific phospholipase C isozymes in the rat nervous system Mitsunori Yamada 1, Masashi Mizuguchi 1, Sue Goo Rhee 2 and Seung U. Kim 1 IDtvtslon of Neurology, Department of Medwme, The Umverstty of Bnttsh Columbta, Vancouver, B C (Canada) and 2 Laboratory of Btochemlstry, Nattonal Heart, Lung, and Blood Institute, Nauonal Instttutes of Health, Bethesda, MD (U S A ) (Accepted 6 November 1990)

Key words Phospholnosltide second-messenger system, Rat, Central nervous system, Immunohlstochemlstry

Developmental changes of 3 phosphomositlde-speclfic phosphohpase C isozymes (PI-PLC-fl, PI-PLC-y and PI-PLC-6) in the rat nervous system were studied by immunohistochemlcal and immunochemlcal methods PI-PLC-y lmmunorcactivity was intensely expressed in the radial fibers from the late fetal to early newborn stages, while weaker PI-PLC-fl reaction was also demonstrated in these structures PI-PLC-fl and PI-PLC-v lmmunoreactlvlty appeared in neurons of various regions after the first postnatal week and then increased to the adult stage Bergmann gha and some astrocytes also showed weak immunoreactivity for both lsozymes from the newborn stage, whde such lmmunoreactive astrocytes were relatively restricted in distnbution m the white matter and hippocampus at the adult stage PI-PLC-6 immunoreactlvlty appeared in astrocytes of entire cerebral regmns from the second postnatal week, although weak antlgenicity was also present in some neurons Immunoblot analysis revealed that the immunoreactlwtles of 3 PI-PLC lsozymes were present in both fetal and adult brains, with strong reactions of PI-PLC-fl and PI-PLC-6 in adult brain and that of PI-PLC-y in fetal brain These results suggest that each PI-PLC isozyme plays important roles in different cell types m the course of their differentiation, and that some PI-PLC isozymes, especially PI-PLC-y, may be involved in cellular division and growth during the histogenesis of the central nervous system the fetal and newborn stages

INTRODUCTION

I n t h i s c o m m u n i c a t i o n , we

report the developmental changes of 3 PI-PLC lsozymes, P h o s p h o m o s m d e - s p e c l f i C p h o s p h o l i p a s e C ( P I - P L C ) is

PI-PLC-fl,

PI-PLC-y

and

PI-PLC-6

a n e s s e n t m l e n z y m e o f t h e s e c o n d - m e s s e n g e r s y s t e m in t h e b r a i n 1°'23'26-28'35 O c c u p a n c y o f r e c e p t o r s by n e u r o -

nomenclature,

transmitters, hormones

by t h e i m m u n o h i s t o c h e m l c a l

PLC

via u n k n o w n

o r g r o w t h f a c t o r s a c t i v a t e s PI-

GTP-bmdmg

protem,

resulting m

f o r m a t i o n o f t w o t y p e s of s e c o n d m e s s e n g e r s , diacylglycerol (DAG)

and lnositol-l,4,5-tnphosphate

phosphatldyhnosttol-4,5-biphosphate

(newly

r e f e r r e d to as P I - P L C - I ,

proposed

PI-PLC-II and

P I - P L C - I I I in p r e v i o u s r e p o r t s a 3 ) , in t h e r a t b r a m s t u d t e d ods with 3 monoclonal ~sozyme 32.

and lmmunoblotting meth-

antibodies

specific

for

each

(IP3), f r o m

(PIP). DAG

stim-

MATERIALS AND METHODS

ulates protein klnase C (PKC), and then phosphorylatlon p r o c e e d s m t h e cell 19 T h e b i n d i n g o f I P 3 to t h e r e c e p t o r s on endoplasmlc retlculum (ER) results m the release of C a 2+ t o t h e c y t o p l a s m f r o m E R 3A3"25_ To d a t e , at l e a s t 5 P I - P L C l s o z y m e s h a v e b e e n i s o l a t e d f r o m d i f f e r e n t s p e c i e s a n d o r g a n s 23, a n d t h e l o c a h z a t l o n o f s o m e l s o z y m e s m s p e o f l c cell t y p e s h a s b e e n r e v e a l e d m a d u l t r a t b r a i n 1'4'24 a n d c u l t u r e s 5 M o r e r e c e n t l y , e a r l y e x p r e s s i o n o f P I - P L C l s o z y m e s h a s b e e n s u g g e s t e d in t h e r a t f e t a l b r a i n b y t h e b ~ o c h e m l c a l TM a n d in sltu h y b n d t z a t l o n s t u d i e s 24

S i n c e ~t h a s also b e e n

known

that

PI-PLC-y may be revolved m cellular division and g r o w t h l 4 15,34 it w o u l d b e w o r t h w i l e a n d i n t e r e s t i n g t o i n v e s t i g a t e w h i c h cell t y p e s s h o w t h e P I - P L C i s o z y m e s in

lmmunohtstochemtstry Wlstar rats pregnant for 17 days (El7) were anesthetized under the inhalation of ether, and rat fetuses were removed from the uteri by Caesarean sections El7 fetuses, newborn rats of postnatal day 1 (P1), day 7 (P7) and day 14 (P14), and adult rats were perfused transcardlally with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde in 0 1 M sodium phosphate buffer, pH 7 4 Following perfuslon the brains of newborns and adult rats were removed from the crama and fixed in the same flxatwe for 18-24 h_ For fetal brains, whole heads were handled in the same way The cerebra and cerebella were cut coronally and sagittally, respectively, and then transferred to PBS for 4 h at 4 °C After washing with graded sucrose of 10%, 20% and 30% m PBS, the tissues were frozen and cut into sections at 10/.tin m thickness by a cryostat The sections were washed with PBS and were immersed in 0 3% hydrogen peroxide m methanol for 1 h at room temperature (RT) to inactivate the endogenous peroxldase activity After washing with

Correspondence S U Klm, Division of Neurology, Department of Medicine, The Umverslty of British Columbia, 2211 Wesbrook Mall, Vancouver, B C V6T lW5, Canada 0165-3806/91/$03 50 © 1991 Elsevier Soence Pubhshers B V (Biomedical Dwlslon)

PBS, the sections were l m m u n o s t a m e d with the av~dnn-bnotm peroxidase complex ( A B C ) method 6 The sections were incubated ]n dilution of monoclonal antubodms against e~ther PI-PLC-fl (9 ,ug/ml), P 1 P L C - / (10 /~g/ml) or PI-PLC-6 (6 ~g/ml) 1i] PBS c o n t a m m g 10% normal horse serum (NHS) for 18 h at 4 °C The ant~ PI-PLC antlbodnes used were mixtures of several monoclonal antnbodles which recogmzed different ep]topes ant~-PI-PLC-fl was a m~xture of clones K 32-3, K - 8 2 3 and K-92-3, anti PI-PLC-/ a m~xturc ol F 7 2 , B-6-4, E 8 - 4 , B-20-3, D 7 3 and E-9-4, ant~ P I P L C - 6 , a mixture of S-II-2, R 2 9 1 , R-39-2 and Z - 7 8 5 a2 The second antnbody was bnotmylated horse ant~-mouse lgG (2 5 ,ug/ml) (Vector Laboratories, B u r l m g a m e , C A ) m PBS c o n t a m m g 10% NHS, and the incubation time was 1 h at R T The sections were then incubated m A B C solution (Vector Laboratories) m PBS for 30 m m at R T Between each step, sectnons were washed thoroughly 3 throes wnth PBS for 1 h Fmally, secttons were incubated m a solution of 50 m M Trns-HCl, p H 7 4, contannmg 300/~g/ml 3,3"-dtamlnobenztd m e t e t r a h y d r o c h l o n d e ( D A B ) and 0 006% hydrogen peroxide

,

~

Some of the adult braun and spmal cord ~ssues were cut nr]to sections by a vtbratome at 21) /~m of thickness The ~nbratomc sections were l m m u n o s t a m e d m a free-floating condition To the first antibodies, 0_25% Trnton X I00 was added, and the mcubatnon t~me of sections m the first and second antibodies and A B C solutnon were 48, 2 and 2 h, respectively Between each step, sections were washed thoroughly with PBS for 1 h by changing the solutnon 3 tnmes At the final step, sectnons were incubated m the D A B solution to vtsuafize the antibody binding sntc,, For negatnve controls, the first annbod]es were adsorbed ~,Lth each PI-PLC antigen or replaced with normal mouse serum

lmmunoblot analysts E l 7 fetal brain and cerebral cortex of adult rat were h o m o g e m z e d nn 5 volumes of ice-cold buffer containing 25 m M Tns-HCI, p H 7 4, 1 m M ethylenedmmmetetraacet~c acnd, ! m M ethyleneglycol b~s N,N,N',N'tetraacet~c acid, 250 m M sucrose, 0 1 m M phenylmethyl sulfonylfluorlde and 0 1 m M dtth~othrentol, and then centnfuged for

4

.r,:,

~, . i l b ~ t l , "

-(

_.'"

-

- - ' :

-

Fig l PI-PLC-fl immunoreactlvlty un the neocortex (A) and cerebellum (B) from E l 7 rat fetus, neocortex of P7 (C), P14 (D) and adult rat (E) A and B weak to m o d e r a t e l m m u n o s t a l n m g Is present in radial fibers V, ventricle; Cbll, cerebellum, CP, chorold plexus C - E PI-PLC Immunoreactlvlty increases m cortncal neurons from P7 to the adult stage Bars• 40 ~ m

L a e m m h ii After S D S - P A G E , the proteins were electrophoretically transferred onto a nitrocellulose paper T h e blots were incubated m 3% bowne serum albumin (BSA) (Sigma) in PBS for 3 h at 37 °C, and then m ddutions of first monoclonal antibodies against etther PI-PLC-fl (18 ,ug/ml), PI-PLC-7 (4/~g/ml) or PI-PLC-6 (10 ug/ml) in PBS containing 3% BSA for 1 h at 37 °C T h e succeeding steps were

20 mln at 10,000 g Proteins in each supernatant were assayed by a Protein A s s a y Kit (Bio-Rad)_ Following addition of equal volume of twofold concentrated L a e m m h sample buffer il and heat treatment at 95 °C for 90 s, 5 p g of protein per each lane were subjected to s o d m m dodecyl sulfate-polyacrylamlde gel electrophoresls (SDSP A G E ) on a 7 5% acrylam~de gel, according to the m e t h o d of

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A

|

•

-

.--

-

e

. " ~ . , ~

i

V

m

Fig 2 PI-PLC-fl immunoreactlvity m the adult rat cerebral white matter (A,B), h i p p o c a m p u s (C), cerebellar cortex (D), anterior horn of the spinal cord (E), p o s t e n o r nerve root (F) and cortlcospinal tract of the brain stem (G) A and B PI-PLC-fl-positive astrocytes are scattered in the white matter E p e n d y m a l cells are also strongly positive. V, ventricle C tmmunoreactlve perlkarya of pyramidal neurons are seen Both the stratum o n e n s (O) and stratum radtatum (R) show weak lmmunoreactivlty D B e r g m a n n gha and dendrites of Purklnle cells stain with anti-PI-PLC-fl antibody. Weak staining is also seen in the molecular layer and the granule cell layer E anterior horn cells show weak immunoreactlvity in their perikarya F: the high level of PI-PLC-fl label is seen at the outermost portions of myelin sheaths G: axons show a m o d e r a t e lmmunostalnlng The outermost portion of myelin (arrowheads) is also stained by the antibody Arrows indicate the nodes of Ranvler Bars, 40/~m

l() done m the same manner as m the staining of cryostat sections The blots were washed thoroughly with PBS containing 0 1% Tween-20 (J T Baker Chemical, Phllhpsburg, N J), and lmmunoreact~on was v~suahzed in the D A B solution

RESULTS

Immunohtstochemtstry PI-PLC-fl zmmunoreactivity. On

El7, PI-PLC-fl im-

munoreactivlty was detected in the radial hbers throughout the brain (Fig. 1A,B). Immature neurons were also positive for PI-PLC-fl, but the staining intensity was much lower than for the radml fibers At the newborn stage, neurons of the neocortex and the basal gangha became weakly positive for PI-PLC-fl and the intensity increased during the development (Fig 1C,D), whde the reactivity of radtal fibers decreased to the undetectable level at P7 After P7, a low level of PI-PLC-fl ~mmuno-

V

~f

m

Fig. 3 PI-PLC-y lmmunoreactlvlty in the neocortex (A), pons (B) and cerebellum (C) from E l 7 rat fetus, cerebellar cortex of PI (D) and adult (E) rats A - C : radml fibers show intense lmmunoreactlvity, V, ventricle; Cbll, cerebellum, CP, chorold plexus D and E the lmmunoreactmvlty is seen in Bergmann gha, external (EG) and internal (IG) granule cells and molecular layer Purkmle cells are mostly negative Bars, 40 ~ m

~

~

N

~

gg'~N

~-

g2~=

eg~

~-~

12

t

L

r

I II

D

,

!

Fig 5 PI-PLC-b ~mmunoreactwnty m developing cerebrum obtained from P7 ( A ) , P14 ( B , C , E ) and adult rats (D F G A intense ~mmunoreactnwty Is seen m the germinal layer (GL), with w e a k staining in scattered cells B,C a m o d e r a t e staining xs seen m astrocytes in the developing whnte matter (B) and brain stem (C) D m the adult white matter, strong lmmunoreactlv]ty IS observed m astrocytes E,F the tmmunoreactnwty increases in astrocytes at the cerebral cortex after P14, wnth a w e a k staining m some neurons G astrocytes m the hnppocampus are pos,tnve for PI-PLC-6 Bars, 4 0 / ~ m

13

reactwlty was detected m some ghal cells, probably astrocytes, in the forebrain and brain stem In the cerebellum, Purkinje cells and Bergmann glia showed weak tmmunoreactlvity. Weak to moderate labeling of PI-PLC-fl was also detected in the choroid plexus epithelial cells and ependymal cells. At the adult stage, PI-PLC-fl lmmunoreactive neurons were scattered throughout the cerebral cortex with vartous intensity (Ftg 1E). Intensely stained neurons were especially located m layers of II, V and IV with marked PI-PLC-fl labehng m the dendrttes and axons as well PI-PLC-fl immunoreactivity was observed m some astrocytes in the cerebral whtte matter, being intense m the cell bodtes and sparse in the processes (Fig 2A,B). In the htppocampus, pyramidal neurons showed the strongest lmmunostainmg for PI-PLC-fl (Fig. 2C). In the cerebellum, PI-PLC-fl ~mmunoreactlvtty was dtstributed m the neuropil of the molecular layer. Bergman glia and dendrites of occasional Purkinle cells were also stained moderately (Fig_ 2D) Granule cell layer showed a weak PI-PLC-fl immunoreactlvity In the spinal cord, PI-PLC-fl immunoreactwe neurons were scattered throughout the gray matter with various intensity (Fig 2E) A high level of immunoreactlvity was observed in the substantia gelattnosa Some astrocytic cells located in the spinal white matter showed moderate antlgentcity Posterior nerve roots showed weak lmmunostainmg m the axons, while mtense ~mmunoreactivity was present at the outer edges of myehn sheaths, probably in the cytoplasm of Schwann cells (Fig 2F) Throughout the experiment, the cytoplasm of oligodendrocytes was negative for PI-PLC-fl; however, some of the outer edges of the central myelin sheaths showed weak but deflmte lmmunoreactlvity for the lsozyme (Fig 2G) PI-PLC-v trnmunoreacttvtty Developmental change of PI-PLC-), lmmunoreactlvity was s~mllar to that of PIPLC-fl; however, the antigenlclty was much more pronounced tn certain structures as described below On El7, PI-PLC-), lmmunoreactlvlty was intensely posltwe m the radml fibers, being clearly traced from the ventncular perlkarya to the subpml endfoot vancoslttes (Ftg 3 A - C ) In the cerebellum, PI-PLC-), antigeniclty m radial fibers became undetectable at P1, while Bergmann gha showed a moderate lmmunoreactivlty for PI-PLC-y in the cell bodies and processes (Ftg. 3D), which was continuously observed untd the adult stage (Fig. 3D). During the cerebellar development, Purkinle cells were not immunostamed by the anti-PI-PLC-7 antibody, whde the external and internal granule cells showed weak tmmunoreactwity. In the cerebral cortex and hlppocampus, a moderate level of staining m radtal fibers could be observed until the P1 stage (Fig. 4A,D), and then the reactivity decreased and disappeared at P14 PI-PLC-y

1

2

3

4

,5.

6

7

Fig 6 Immunoblot of rat fetal (lanes 1,3,5) and adult (lanes 2,4,6) brain extracts with antl-PI-PLC-fl (lanes 1,2), antl-PI-PLC-7(lanes 3,4) and antl-PI-PLC-6 (lanes 5,6) antibo&es Molecular weight standards (97, 66, 43 and 31 kDa) are shown on lane 7 Both extracts yield posltwe bands for each PI-PLC lsozyme The staining levels of PI-PLC-fland PI-PLC-6 are stronger in adults (lanes 2,6), whde that of PI-PLC-v is in the fetus (lane 3)

immunoreactwtty m neurons appeared at the P7 stage and increased gradually according to the development (Fig 4B,C,E,F) Compared to the results of PI-PLC-fl staining, PI-PLCw immunoreactwtty was more prevalent m cortical neurons and was much stronger in axons (Fig. 4G,H). At the P7 and P14 stages, many bipolar or multtpolar cells posittve for PI-PLC-), could be observed throughout the brain Such cells were not detectable any more in the adult stage, whale PI-PLC-y lmmunoreactlve astrocytes were scattered in the cerebral white matter and hippocampus. Although cell bodies of ohgodendrocytes seemed to be negative for PI-PLC-7, the outer edges of myehn sheaths were ~mmunostamed circularly with antiPI-PLC-fl antibody (Fig 4G), hke the results of PIPLC-fl staining Such a staining pattern of myelin was also observed m the posterior nerve roots (Fig. 4H)_ Throughout development, chorold plexus eplthehal cells and endothelial cells were positive for PI-PLC-7 PI-PLC-t~ immunoreactivity. On El7, no spectftc regions or structures were selecttvely stained with antiPI-PLC-6 antibody, while a relatively low level of PI-PLC-6 label was present throughout the bram On P1, ventncular germinal cells and chorold plexus eplthehal

14 cells showed weak lmmunoreactivity After P7, bipolar or muitlpolar cells, probable immature astrocytes, became positive for PI-PLC-b (Fig 5A-C,E). These cells were distributed throughout the brain_ At the adult stage, astrocytes showed intense immunoreactivlty for PI-PLC6 (Fig 5D,F,G), while a low level of PI-PLC-b label was also recognized in some neurons (Fig 5G,H) In the control sections, no positive staining was observed Immunoblot analysts. Extracts from the El7 brain and cerebral cortex of adult rat yielded protein bands corresponding to all of the 3 PI-PLC Isozymes, PI-PLC-fl (150 kDa), PI-PLC-7 (145 kDa) and PI-PLC-0 (85 kDa) (Fig. 6) The antigenlclty of PI-PLC-fl and PI-PLC-6 was more concentrated in the adult rat cerebral cortex, while that of P1-PLC-y was more in the fetal brain DISCUSSION These results indicate that the lmmunoreactlvity of 3 PI-PLC isozymes is expressed in the rat fetal brain from as early as E17, and that developmental changes of 3 Isozymes occur in different cell types in different ways P1-PLC-fl and PI-PLC-),, especially the latter, were strongly expressed in the radial fibers durmg late fetal and newborn stages. It is possible that some processes of developing neurons might be Included in the lmmunoreactive radial fibers in the tissues of E17 However, predominant presence of PI-PLC-positlve radial fibers which could be traceable from ventrlcular wall to subplal region even In the newborn stages suggests their radxal gila origin, because neurons had finished their migration to the cortex. Although it is speculated that radial processes of the radial glia nerve serve as guides for neuroblast migration In a developing brain 2,2°-22, the biology of the cells has not yet been clarified The present study would show one of the functional aspects of radial gha, particularly on the slgnal-transductlon system In this cell type It has been reported that a primary structure of the PI-PLC-~, isozyme has a significant sequence similarity with the src-encoded family of tyrosine klnases z3, and that tyroslne phosphorylation of proteins would be implicated both in mitogenlc signal pathways and in oncogenlc transformation 9. It is also known that there is a variety of receptors which act via PI-PLC and phosphoinositIde pathways Among such receptor-mediated mechanisms, growth factor receptors such as those for epidermal growth factor and platelet-derived growth factor could be involved 14'15'34 Thus the PI-PLC-y isozyme in radial gha may be related to cellular division and growth of this cell type in the course of histogenesis of the central nervous system (CNS) mediated by such growth factors

PI-PLC-fl and PI-PLC-y Immunoreactlvlty appeared in neurons of the cerebral cortex at 177 and increased thereafter according to the development The distribution and intensity of this reactivity m the adult rat cerebrum were almost identical to the previous study 4 The present study offers more information regarding the distribution of PI-PLC-fl and PI-PLC-v. In the cerebellum, PI-PLC-fl lmmunoreactlvlty was detected in some Purklnje cells after P7, and the reactivity increased to moderate level at the adult stage. PI-PLC-y lmmunoreaction was negative in this cell type Granule cells were weakly positive for PI-PLC-/3 and PI-PLC-y, and the molecular layer was moderately immunostalned. Bergmann gha showed a moderate Immunoreactivlty for both lsozymes throughout the development The intensity of immunoreactivlty in cerebellum is considerably less than that in cerebrum In this regard, it has been reported that the cerebellum contains high amounts of another type of PI-PLC l s o z y m e 24 PKC and IP 3 receptor are molecules which would function in cooperation with PI-PLC in the phospholnosltide second-messenger system23'3~; however, neither distribution of PKC 7's 2v nor IP 3,13,25 receptor matches exactly with those of PLC isozymes shown in our results In the present study, lmmunoblot analysis revealed the existence of PI-PLC-6 in the rat fetal brain, although specific localization of the lsozyme could not be demonstrated by immunostainlng. After the first postnatal week, PI-PLC-6 immunoreactivity increased gradually in astrocytlc somata and processes which eventually were distributed throughout the brain at the adult stage. PI-PLC-b is expressed In astrocytes at approximately the same time when radial glias are transformed into astrocytes and ghal flbrillary acidic protein (GFAP) is expressed in this cell type 16'33 PI-PLC-fl and PI-PLC-y immunoreactlvity was also detected in astrocytes in the adult rat brain, however, such lmmunoreactive astrocytes were dominantly distributed in the paraventricular region such as the cerebral white matter In the mature brain, astrocytes in different regions contain different levels of GFAP, with higher amounts found in white matter than gray matter 1~ In addition, astrocytes regulate GFAP m R N A level by cyclic AMP and PKC-dependent mechanisms 3~. Taken together with the results of our observations, PI-PLC-fl and PI-PLC-y may be involved in the regulation of GFAP expression in astrocytes. Somata of ohgodendrocytes were negative for any PI-PLC isozymes, however, circular stamings of PIPLC-fl and PI-PLC-y were detected at the outer edges of myelin sheaths, implying specific locahzatton of P1-PLC enzyme on ohgodendrocytic processes. Such a staining pattern was also observed in the spinal posterior nerve

15

presence of phosphoinostttde second-mes-

P I - P L C - y to P I - P L C - 6 d u r m g the postnatal d e v e l o p m e n t .

s e n g e r s y s t e m m o l t g o d e n d r o c y t e s h a s also b e e n sug-

Similar c h a n g e s m e x p r e s s i o n o f d i f f e r e n t i s o e n z y m e s in

gested by a study on the distrtbutton of P I - P L C tsozymes

d i f f e r e n t C N S cell t y p e s h a v e also b e e n r e p o r t e d m the

m C N S cell c u l t u r e s 17

d e v e l o p m e n t a l p r o f i l e s o f e n o l a s e t s o z y m e s 3°

roots. The

In c o n c l u s i o n , o u r r e s u l t s s u g g e s t t h a t t h e t m m u n o r e activity o f P I - P L C - f l a n d P I - P L C - ) , i n c r e a s e s m n e u r o n s as t h e y d i f f e r e n t i a t e p o s t n a t a l l y , a n d t h a t P I - P L C - v ts s t r o n g l y e x p r e s s e d in r a d i a l glia by the n e o n a t a l s t a g e , but PI-PLC-b becomes dommant thereafter m astrocytes T h u s it is e v i d e n t t h a t a s t r o c y t e s u n d e r g o a switch f r o m

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Acknowledgements This work was supported by grants from the Medical Research Council of Canada and the Multiple Sclerosis Society of Canada We thank Myong Klm and David Osborne for their technical assistance, and Kathleen Stanfleld for her secretarial assistance

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