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A . M . R O B E R T S O N , R . H . \ I . K ( N G . J . R. M I D D L E A N D P . K. T H O M A S

D ep a rtm en t o f C linical Seuroscien ces. R oyal Free H ospital S chool of M edicine. London. i'K

(.A ccepted 25 S nvem h er 1996)

a b s t r a c t

T h e T rem bler-J ( T r ' ) m o u se has a poin t m u tation in the gen e c o d in g for peripheral m yelin p ro tein 22 (P M P 2 2 ). D istu rb an ces in P M P 22 are associated w ith a b n orm al m y elin a tio n in a range o f inh erited peripheral n eu rop ath ies b oth in m ice and hum ans. P M P 22 is p rod u ced m ain ly by S chw ann cells in the peripheral nervou s system w here it is localised to co m p a ct m yelin . T h e fu n ction o f P M P 22 is u n clear but its lo w a b u n d a n ce 5% o f total m yelin protein ) m eans th at it is u n lik ely to play a structural role. Its in clu sio n in a recently d iscovered fam ily o f proteins su ggests a fu n ctio n in cell p r o lifer a tio n /d iffer e n tia tio n an d p o ssib ly in a d h esio n . N erves from T r ' and the allelic T rem b ler (T r) m ou se are ch aracterised by a b n o rm a lly thin m yelin for the size o f the a.xon and an in creased n u m ber o f Schw ann cells. W e report ultrastructu ral ev id en ce o f abn orm al Schw ann cell-a.xon in tera ctio n s. S ch w an n cell nuclei h a v e been fo u n d a d jacen t to the n od es o f R an vier w hereas in norm al an im als th ey are loca ted near the cen tre o f the in tern o d es. In so m e fibres the term inal m yelin lo o p s faced o u tw a rd s in to the extracellular sp a ce instead o f tu rn ing inw ards and term in atin g on the a.xon. In severely affected nerves m an y axon s were on ly partially su rro u n d ed by S ch w an n cell cytop lasm . A ll these features su g g est a failure o f Schw ann c ell-a .x o n r eco g n itio n o r in tera ctio n . In a d d itio n to abn orm alities related to ab n o rm a l m y elin a tio n there was sig n ifica n t a x o n a l lo ss in the dorsal roots.

K e y w o rd s: P e r i p h e r a l n e r v e ; m y e l i n a t i o n ; h y p o m y e lin a tio n . S c h v ,a n n c e ils ; p e r ip h e r a l m y e lin p r o t e i n 22; T r e m b le r - J m o u s e .

I N T R O D t j 'C T I O N

is in a p p ro p ria tely thin for the size o f the a.xon (L o w , 1 9 7 6 a , b , 1977) and has a ten d en cy to be u n co m - T h e Trem bler-J m o u se ( T r ' ) w as discovered by p a cted . esp ecia lly near the n od es o f R a n v ier (A yers <St S id m an et al. (1979) w ith in a co lo n y o f C 5 7 B L /6 J A n d e r so n , 1973, 1975; L ow . 1977). B oth T r ’ and Tr

m ice. A t a p p ro x im a tely 3 wk o f age. affected anim als have been fo u n d to be associated w ith p o in t m u ta tio n s c o u ld be d istin gu ish ed from their nonaffected litter- in the gen e c o d in g for peripheral m yelin p ro tein 22 m a tes by an a ctio n trem or an d gait abnorm ality. T he (P M P 2 2 ) (S u ter et al. 1992a. 6). T h e P M P 2 2 gen e latter in v o lv ed prim arily the hin dlim bs and con sisted e n c o d e s an 18 k D a protein w ith 4 p u ta tiv e trans- o f e x te n sio n o f the kn ees an d an k les and splaying o f m em b ran e d o m a in s and 1 N -lin k ed g ly c o sy la tio n site the h in d lim b s (H en ry et al. 1983). On the basis o f (.M an fioletti et al. 1990; W elcher et al. 1991; Su ter et g en etic an d m o rp h o lo g ica l stu d ies T /"'w as ju dged to al. 199 2 a ). T h e resulting 22 k D a g ly c o p r o te in is be a m u ta tio n at the sam e locu s as Trem bler (T r) p ro d u ced in the peripheral n ervou s sy stem m ain ly by (S id m a n et al. 1979). a n eu ro lo g ica l m ou se m utant S ch w a n n cells w here it is lo ca lised in the co m p a c t d isco v ered by F a lco n er (1951). P a th o lo g y in both m y elin o f essen tia lly all m yelin ated fibres (S n ip es et al. strains o f m ice is con fin ed to the peripheral nervous 1992). Tr ’ has a poin t m u tation w h ich rep laces a system and is ch aracterised by a m yelin deficit and an leu cin e w ith a p rolin e residue at a m in o acid p o sitio n increased nu m ber o f S ch w an n cells (.Ayers & 16 (S u ter et al. 1992a). The 7"r 'm u t a t io n is in the 1st .A nderson, 1973; L ow & M cL eo d , 1975; A gu ayo et tra n sm em b ra n e d om ain o f P.M P22 w h erea s the ab- al. 1977; Perkins et al. 1981). M yelin in these anim als n orm ality in Tr is the replacem ent o f a g ly cin e by an

C o r r e s p o n d e n c e to .A n d re a R o b e r t s o n . D e p a r t m e n t o t 'C li n i c a l N e u r o s c ie n c e s . R o v a l F r e e H o s p ita l S c h o o l o f M e d ic in e . R o w l a n d H ill S tr e e t. L o n d o n N W J 2 P F . L K .

aspartic acid residue at am in o acid p o sitio n I5() in the 4th transm em brane region (S uter et al. 14^2/)). A lth o u g h m ost strongly e.xpressed in peripheral nerve. P M P 22 m R N A is (bund in oth er adult tissues in clu d in g lung, co lo n and brain (S preyer et al. 1991 ; W elcher et al. 1991; Bosse et al. 1994: Suter et al. 1994). PM P 22 mRN.-X is a lso e.xpressed in early o rg a n o g en esis in the gut and su rrou ndin g the liver c a p su le, and later in several m esoderm derived co n n e c tiv e tissues and ectod erm derived cells ot' the lens and in the skin (B aech ner et al. 1995). Its w idespread expression in oth er tissues suggests a role for P.MP22 ou tsid e the nervous system . The gene for P M P 22 was fou nd to be identical to the grow th arrest specific gene, g a s 3 (Spreyer et al. 1 9 9 1 ; W elcher et al. 1991) which belongs to a grou p o f genes w hose ex p ressio n is specifically a sso cia ted w ith the quiescent cell state and w hich m ay be in v o lv ed in the regulation o f general cell grow th (M a n fio letti et al. 1990; ZoidI et al. 1995). P a th ological ch an ges in m ice w ith alter­ a tio n s in P M P 22 are con fin ed specifically to the peripheral nervous system . In P M P 22 deficient m ice the o n set o f m yelin ation is d elayed , follow ed by h yp erm yelin ation and su b seq u en t dem yelin ation (A d lk o fn e r et al. 1995). T h is su ggests that PM P22 fu n ctio n s in the initial stages o f m yelin form ation and later in co n tro llin g m yelin th ick n ess and stability. U ltrastru ctu rally. P M P 22 im m u n oreactivity has been lo ca lised to regions o f co m p a ct m yelin and was not d etected over the regions o f the in tern od es which co n ta in the u n com p acted S ch m id t-L a n term a n incisures (S nipes et al. 1992). P.MP22 has also been lo ca lised on the p lasm a m em b ran e o f S chw ann cells o f R em ak fibres, the m em b ran es o f Schw ann cell p ro cesses that surround co lla g en p ock ets and S ch w a n n cell plasm a m em b ran es in the cell lam inae o f o n io n bulbs in the nerves o f type LA C h arcot-M arie- T o o th disease ( C M T lA ) (H a n ey et al. 1996). This su g g ests that all Sch w an n cells, n o t o n ly those in v o lv ed in the m yelin ation p rocess, con stitu tively exp ress PM P 22.

T h e recent d iscovery o f a novel gen e fam ily o f structurally related proteins o f w hich PM P 22 is a m em b er provid es the stron gest in d ica tio n s o f the gen eralised p h y sio lo g ica l role o f P M P 22 (T aylor et al. 1995). T he 4 proteins so far discovered . E.MP-I (T a y lo r et al, 1995). M P 30 (K u m a r et al. 1993). C L 20 (M arvin et al. 1995) and P M P 22 (S n ip es et al. 1992) each have 4 pu tative transm em brane d om ain s with a co n serv ed N -lin k ed g ly c o sy la tio n site on the 2nd transm em brane d om ain . T h e high degree o f identity, particularly o f the first 2 transm em brane dom ains, su g g ests they m ay serve a sim ilar and im portant

fu n ctio n . T he oth er m em b ers o f this protein fam ily apart from P.MP22 are fou n d in d ilferen tiatin g and m ature cells rather than being a sso c ia te d w ith grow th arrest. T h e fu n ction o f this fam ily m ay be related both to the sw itch from p roliferation and to the m a in ­ tenan ce o f critical fu n ction s in the differentiated state (T a y lo r et al. 1995). In the present stu d y, the m o rp h o lo g ica l ch an ges in the peripheral nerves have been exam in ed in the Tr ' m o u se to seek in d ication s as to the m ech an ism s o p erative in the d ev elo p m en t o f the n europathy.

M E T H O D S

M ale and fem ale affected ( T r ' f - > r ) and un affected

{ + / + ) litterm ates were used at 3 an d 12 m o o f age. A n im a ls were killed by p erfu sio n w h ile under d eep a n a esth esia (S agatal 18 m g /a n im a l). T h e th orax w as o p en ed and a butterfly n eed le a tta ch ed to a can n u la inserted in to the left ventricle o f the heart. T he right atrium w as incised and b lo o d w a sh ed ou t w ith 1 N sa lin e at 37 °C. T his w as fo llo w e d by perfusion w ith

1 % p araform ald eh yd e and 1 % g lu tarald eh yd e in 0.1 M P IP E S (p ip era z in e-N .N '-b is-[2 -e th a n esu lfo n ic acid]) buffer (p H 7.4). in itially at 37 °C and then at r o o m tem p eratu re. T he sciatic nerve w as rem oved and laid on p ieces o f card w hich w ere then placed in fresh fixative. T h e dorsal and ventral ro o ts w ere excised to g eth er w ith the dorsal root g a n g lia . T he lum bosacral sp in al cord w as also rem oved . A ll tissue w as then fixed for a further 2 h. w ash ed in P IP E S buffer plus 2 %

su cro se an d then postft.xed in 1 % o sm iu m tetroxide in P IP E S buffer plus 2 % su crose. 3 % so d iu m iod ate and 1.5% p o ta ssiu m ferricyan ide. T h e specim en s w ere d eh yd rated in increasin g c o n c en tr a tio n s o f eth a n o l a n d transferred to e p o x y resin via 1.2-epo.xypropane as an interm ediary. S em ith in sec tio n s w ere cut and sta in ed w ith th ionin.

M ea su rem en ts o f a x o n d ia m eter, fibre d iam eter and g ratio (a x o n d ia m e te r /to ta l fibre diam eter) were c o n fin ed to fibres w ith a o n e -t o -o n e a x o n /S c h w a n n cell rela tio n sh ip ; these in clu d ed am yelin ate or d em y elin a ted fibres. T he n u m b ers o f Sch w an n cell n u clei in ea ch sectio n were o b ta in e d from transverse sectio n s th rou gh the dorsal a n d ventral spinal roots. .All m easu rem en ts were tak en from w h ole fascicles view ed w ith a x 100 ob jective a n d a x 1.2 op tiv a r on a Z eiss A x io p la n m icro sco p e (Z eiss U K . W elw yn G ard en C ity. H erts. U K ) c o n n e c te d on -lin e th rou gh a telev isio n cam era to a K o n tro n I B AS .AT im age an alyser (Im agin g A sso c ia te s. T h a m e . U K ). M any fibres in the T r ' an im als had m yelin that w as to o thin to be identified (less than 5 m v elin lam ellae) at the