Ovine ceroid lipofuscinosis : a thesis presented in partial fulfilment (70%) of the requirements for the degree of Master of Veterinary Science at Massey University

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(2) OVINE CEROID-LIPOFUSCINOSIS. A thesis presented in partial fulfilment of the requirements for the degree of Master of Veterinary Science at Massey University. Antonie Janrnaat. 1979. (70%).

(3) to. my. pare n t s ... "Ie hebt. 't good edo"'an "... ..

(4) ABSTRACT. Ov ine ceroid-l ipofu scino s i s i s a rare inh e ri ted neu rological di se a s e which ha s only been di agnosed in one fami l y of the South Hampsh i re breed o f sheep .. Th i s breed i s o f re la tive recent origin a nd was e s tab l i shed. from a n initia l cro s s between Sou thdcwn and Hampsh ire Down sheep .. Af fected. sheep show loss of vi s ion and behav iou ra l abnorma l i t i e s starting at 1 1 - 1 2 month s o f age , wi th motor dysfunc tion commenc ing soon a fterwa rds.. The. c l i n i c a l s i gns inc r e a s e in sever ity a s the d i s e a s e progre s s e s and unde r field con d i t ions a f fected anima l s are not expected to l ive b eyond 2 years .. Ovine ceroid-l ipo fu scino s i s i s cha racte ri zed h i s tologica l ly by the i ntracytopl a smic accumu la tion of PAS and Sudan b l ack po si tive autof luorescent l ipop igment ma te r i a l in neuron s and a wide variety of other c e l l type s .. The proce s s l eading to the accumu l a t ion o f l ipopigme n t. seems o n l y t o dama g e n eurons a nd there i s degenera tion a n d l o s s o f neurons , e specia l ly in the cerebra l cortex a nd the v i sua l neuroepi the l i um o f the re t ina i . e . retina l a trophy . reduc t i o n in s i z e a nd we i gh. Gro s s l y , a f fected bra ins show. on average 66% of those of norma l sheep .. U l t ra s tructura l l y , the typ ica l l ipop i gment inc l u s ion i s a round or ova l body 0 . 2 - 5 . 0 �m in s i ze , o f va rying e l ectron density, a wide �ariety of memb ranous profi l e s may b e seen .. in which. Some o f the. (. memb ranous patterns have rece ived spec i a l names such as curvi l i n e a r , finge rpr int a n d crysta l l o i d ..

(5) Pathological examinat ion of l i ver , skin and recta l biopsy mate r i a l o f l ambs a t 4. -. 5. mon ths o f age shows the pre sence o f ac cumul ated. l ipopigment , and i s a means of early d i agno s i s be fore the onse t o f c l i n i cal s i gn s .. Thi s obs e r va t i on and the fa c t that l ipopigment h a s be e n. demon s t ra ted in a f fe c t ed lambs a t bi rth , show ovine cero i d - l ipofu s c i no s i s to be a s soc iated w i th a true inborn e rror o f me taboli sm .. The fami ly tree o f a l l a f fe c ted sheep and the resul t s o f s i re­ daughte r mat ings of a hete rozygou s ram show the d i se a se to be inh e r i t e d a s a s impl e au to soma l rece s s ive tra i t .. The d e l e t e rious gene for ovi ne. c e roid-l ipofusc ino s i s i s u n l ike ly to b e of economic importance to the sheep industry a s the Sou t h Hampsh i re breed was developed to suppl y s i r e s for termina l cros se. s a s soc iated with table l amb produc t ion .. Th e obj e c t s of this study were to d e f i n e ov ine ceroid-l ipofu s c i no s i s i n c l inic a l , patholog i c a l a n d genetic te rms , and to compa re i t w i th s im i l a r d i se a s e s in man and dome s t i c a n ima l s .. I t is conc l uded that the. ov ine di sease doe s indeed be l ong to the hete rogeneou s g roup o f d i s e a s e s o f ma n and dome s t i c anima l s known a s Batten ' s d i s ease o r the neuron a l o r genera l i sed c ero i d - l ipo fu s c i no se s .. Of th e s e the ovine e n t i ty mo s t. c l o s e l y resembl e s the l a t e i n fant i l e and the j uvenile forms of the human synd rome , and the can ine disea se .. I t i s proposed that ovine c e ro i d -. l i po fu sc ino s i s woul d make a u s e fu l e xperimenta l mode l for Batten ' s d i s e a se ..

(6) ACKNOWL E DGEMENTS. This the s i s re f l ec t s a substantial part o f the rese arch I d id wh i l e employed as a Re sea rch O f f ic e r in the Depa rtment o f Ve te rina ry Patho logy and P ub l i c Hea l th a t rla s s ey Un iver s i ty ove r a per iod of 3 yea rs . I am g ra te fu l to Prof . B . W . Manktelow , as Head o f Depa r tmen t , and to Dr R . D . Jo l l y ,. as leader o f th e r e s earch team , f i r s t l y for a l lowing me. to u s e the r e s e arch find i n g s i n th i s the s i s and second l y for the pert i nent advi c e , pa r t i cu l a r l y Dr Jo l l y ' s , pro ffered in the i r r ol e s a s supervi sor in i t s prepa ra t ion .. A number o f peop l e gave pro f e s s ional. advic e an d technical a s s i s t a nce du ring th e course o f my s tudy i nto ovine c e r o i d - l ipofusc inos i s .. Mr. D.M . We s t gave generou s l y o f h i s time w i th the c l i n i c a l. exam i na t i on o f the anima l s a n d supervised the an a e s thet ic , surg ical and po s t-operat ive procedures i nvolved in the taking o f sk i n , l iver and r e c ta l biops i e s .. He a l so suppl i e d the norma l retinograph. fi gure 2 . 3 as a contro l . haemogram e st imation s .. u se d in. Dr R . H . Sutton and h i s s t a f f performed the Ms . K . C a to took the radiog raph s wh ich we re. inte rpreted b y Dr M. O ' Ca l l agha n .. Prepa r a t ion o f t i s su e s for l ight and f luore scent mic ro scopy was c a r r i ed out by Mr C. Fletche r , Mr R . P . Han sen , Ms . S . L . Ma l l och a nd Mr s P . M . S l a c k . coope rat ion .. I thank them for the i r cheerful and f r i endly. Mr A.S. C ra ig and Mr D . Hopcroft o f the Departmen t o f. S c i e n ti f i c a n d Indu s t r i a l Research gave very helpfu l a dvice on the preparation of t i s sues for e le c t ronmicro scopy and printed the e l ec t ronmic rograph s .. M r T. Law mos t obl igingly prin te d the o th e r.

(7) photographs used i n th i s the s i s .. I am i n debted to Mrs E . Br i s tol. and Al i s o n for typi n g the f i r s t draft of the manu script , and to Mr s F . S . Wicherts for typ i ng the f i na l copy .. Fina l l y ,. I wou ld l ike to thank the farm and hosp i t a l techn i c a l. s taff for the ir care o f and i n terest i n the anima l s , and espec i a l l y Mr. A . T . DeCl eene for hi s cheerful and knowl edgeable a s s i s tance w i th. the handl i n g o f the sheep i n the f i e l d , hosp ital and post-mortem room .. The research pro j ec t i n which I was i nvolved w a s supporte d by the U n i t e d S tates Na tion a l In s t i tutes o f Health Gra n t No . RO l NS 1 1 2 3 8 - 0 4 and 5 from the National I n s t i tute of Neuro logic a l Di sord ers and S troke ..

(8) TABLE OF C O NT E NTS. Page LIST OF TABLES. LIST OF FIGURES. INTRODUCTION. 1. CHAPTE R I :. Review - the neurona l ceroid - l ipofu s c i nose s .. CHAPTER I I :. C l inica l f indings .. 43. CHAPTE R I I I :. Pathology .. 63. CHAPTER IV :. Genetic s .. 104. Ge ne r a l d i sc u s s ion .. 109. C HAPTER. V:. REFERENCES. 2. 115.

(9) LIST. OF. TABL E S. Table. Page. l. I. Cl a s s i f icat ion o f the neuronal c e roid -l ipofu sc ino s e s .. 2.I. C a s e h i s tories of Sou th H ampshire sheep af fected with c ero i d - l ipofu sc i no s i s.. 3.I. 64. Di stribut ion o f l ipop i gment i n ceroid - l ipo fu s c i nos i s o f sheep over 1 2 mon ths o f age .. 4.I. 46. L i s t o f South Hamp s h i r e s heep wi th ceroid­ l ipofu s c i no s i s that we r e examined pathologic a l ly .. 3. II. 9. 71. Re su l t s o f s i re -daughter ma t ings of a South H ampshire ram hetero zygous for c eroid- l ipo fu sc i no s i s .. 107.

(10) LIST. OF. FIGURES. Figu re. 2.1. Page. South Hampshire ram , 2 4 months of age ,. 52. a f fected w i th c e roid-l ipo fu s c i nosi s .. 2.2. Graham-Knol l techn iqu e to d emon strate perox ida s e act ivity i n sheep neutroph i l s.. A part i a l ly reacted. a nd a comp l ete l y reacted c e l l a re shown .. 2.3. 55. Retinog raphs o f the ovi ne eye showing attenuat ion and stra ighten i ng o f retina l ve s s e l s in an a f fected 2 4 months old ram , when compa red to a control sheep .. 3.1. 57. Dor s a l v i ew o f bra ins f rom two 1 3 months old ewe hoggets .. The b ra in o f the a f fected sheep i s reduced. in s i ze and shows thinning o f the c e rebral gyri .. The. b ra i n we ight of the a f fe c ted animal is 5 7 . 2 g , compa red with 8 6 . 2 g for the norm a l control she ep .. 3.2. 67. Late ral v iew o f brains f rom two 1 3 months o l d ewe hoggets .. The ce rebrum o f the a f fe cted an ima l shows. reduct ion in s i ze , th i nn i ng of the ce rebra l gyr i and a s l i ght dor so-ventra l f lattening , when compared with the b ra i n o f the normal control sheep .. 68.

(11) Page. Figure. 3. 3. Transverse s e c t ion o f the cerebrum of an a f fected 1 3 month s old ewe hogget and of an age and sex­ matched control she ep .. No te r educ ed size o f c e rebrum ,. ma rked thi n n ing o f cortex, th i nning o f corpus callosum and sepb� pellucidum and e n l a rgement of late r a l vent r i c l e s i n the a f f e c t e d sheep .. 3.4. 69. Cerebral cortex of a 1 5 month s o l d South Hampshi re ewe hogget wi th ce ro i d - l ipofu scinosis showing neuron s l aden with l ipop i gme n t granul es .. 3.5. 73. Tha lamus o f a 2 5 mon ths o l d a f fected ram. Lipopigment granu l e s s ta i n bl ack and in some neurons appe a r to almo st compl e t e l y f i l l the cytopla sm .. 3.6. 73. Cerebra l co rtex of a 2 5 mon ths o l d a f fe cted ram . The whi te s t ruc ture s are a u tofluore scent l ipopigme n t granul e s , s i n g l e o r in cluste r s .. 3 .7. 75. Cerebel l a r cortex o f a 2 5 months old affected ram .. Inclu s ion s in a Purk i n j e ce l l are nume rous. and show va riation in s i ze .. 75.

(12) Page. F i gure. 3. 8. Cerebral cort e x o f a 2 5 month s old ram with c ero i d - l ipofusc inosi s , show ing a neuron surrounded by an increased number of gl i al c e l l s .. Astrocyte s. predominate and may show unusu a l l y shaped nuc l e i.. 3.9. Cerebra l cor t i c e s of two approx imat e l y old South Hampsh ire ewe hogge t s .. 15. 77. mon ths. The affected. an imal shows an incre ase in number and size of a s trocyte s , compare d to the control sheep .. 3 . 10. 77. The h i s tology of the re t i na o f a n 18 months old South Hampsh i re ewe w i th ceroi d -lipo fuscinos i s , compared w i th that o f a norma l sheep .. The a f fected. an ima l shows severe atrophy o f the l ayer o f rod s '. a nd cone s and the outer nuc l e a r l ayer .. The re t i n a l. p igment ep i the l ium , i nner nucl ear l ayer and gangl ion cel l l ayer appear normal .. 3 . 11. Lymphocyte s from a 25 months o l d a f fected ram . Note absence o f l ymphocytic vacuolat ion .. 3 . 12. 78. 84. Re sidual body i n cort i c a l n euron showing l obul ated shape .. 84.

(13) Page. Figur e. 3 . 13. Storage material appare ntly free in the cytoplasm o f a men inge al cap i l lary endothelial c ell .. Shor t. arrow po ints t o a five - l ayered membrane , wh ich a t long a rrow seems to b e formed b y the fu sion o f 85. two tr iparti te membrane s .. 3 . 14. Part o f l i popigment body i n ret in al neuron showing granu l a r matrix of vary ing e l ec t ron density , and five - l ayered membrane s .. The arrow ind i cate s a 85. mye l i n pattern .. 3 . 15. P a r t o f res i dual body i n a ventral horn neuron showing stacks of alte rna t ing dense and l ight l ine s , with a period i c i ty of approx ima te ly 5 . 2nm .. 3 . 16. Panc rea t i c acinar c e l l w i th curv i l i near inc lus ion bod i e s .. 3.17. The cellula r a rch i tecture appears no rma l .. 88. Storage body w i thin a c e reb r a l c o r t i c a l neuron showing membranous pro f i l e s and tubu l ar a rray s .. 3.19. 87. I nclu s i on i n c e rebral cor t i c a l n euron showing f i n gerprint and c rysta lloid pattern s .. 3.18. 87. 88. Part o f ventral horn neuron o f lumbar cord showing a l a rge numb e r o f inc l u s ion s .. The c ytop l a sm shows. spa r s ity of norma l organe l l e s .. 89.

(14) F i gure. 3 . 20. Page. Membranous inclus ion body in c e l l a t the cort ico­ medu l l ary j unction of the adrenal g l and .. 3 . 21. 89. Membranous inc l u s ion in a thick bun d l e of a s trocyte fibre s in the cerebra l cortex of a 25 months old ram with ceroi d-l ipo fuscino s i s .. 3 . 22. 91. Pancre a t i c ac inar c e l l showing two type s of residua l bodi e s .. A curv i l inear body typical for. ovine ceroid-l ipo fu s c ino s i s , and a granul ar , more e l ec tron den se , body wh i ch may a l so be seen in norma l sheep .. 3 . 23. 91. Lymphoid fo l l ic l e in rec ta l wa l l of a 5 months old ewe l amb .. Ma cropha g e s cont a i n nuc lear remnants. a nd l ipopigment inc l u s i on s. 3 . 24. 94. .. Ultra s truc tural deta i l o f part o f m a crophage showing typic a l curvi l i near bod ie s .. Thi n sec tion. obtained from s ame t i ssue a s shown in figure 3 . 2 3 .. 3 . 25. 94. Granular electron dense inclusion in a m acrophage in the rectal wall of a 5 months old normal lamb. These inclusions are encountered in both normal and affected sheep, and carry no significance in the diagnosis of ovine ceroid-lipofuscinosis.. 95.

(15) Page. Fi gure. 3 . 24. Autofluore scent ma t e r i a l ou t l i n e s the sweat g l a nds i n the sk i n of a 25 months o l d ram affected w i th ceroid-l i pofusc ino s i s .. Before the on set of c l i n i ca l. s i gn s , th i s autofl uore scence i s a l re a dy pre sent i n affected l amb s a t 4. 4.1.. -. 5 months o f a ge .. Fam i l y tree of affected i n d i vi dua l s .. 95. On ly ma t i n g s. o f heterozygot e s a n d othe r pertinent i ndividua l s a re shown .. 106.

(16) 1. I NTRO DUCT I ON. The neuronal cero i d -l ipo fuscino se s , also known a s the non - i n fan tile amaurotic family idioc i e s and as Batt en ' s dis e a se , are a d i s t inct group They are characte ri sed by the. o f i nheri ted d i se a s e s o f children .. pro gre s s i ve intracel l ular , and e speci ally in traneuron al , accumulat ion of the a uto fluore scent l ipopigments , ceroid and lipo fuscin .. A f fected. patients show progre s s ive central nervous sys tem de gen e rat ion , lo s s o f v i s ion , l o s s o f mental facult ie s , s e i zure s and premature death . An adu l t form, w i th mi l der symptomatology i s also recogn i sed .. S imilar. d i s e a s e s have also been de scribed in a number of spec i e s of dome s tic animals .. The d i sease a s i t occurs in the South Hampshire sheep fo rms. the sub j ect ma teri al o f th i s the s i s .. The neuronal ce ro i d -l ipo fuscino s e s o f man and dome stic an imal s are reviewed i n Chapter 1, wi th empha s i s on c l i n ical , pathologica l , biochemical and gene tic findings .. A bri e f hi s torical account i s a l so. pre s ented in an a ttempt to cl a r i fy the con fus i n g nomencl ature e xtant in th e l i tera ture , by tracing the origins of the various eponymic and descript ive n ame s .. The ma i n ob j ectives o f the s tudy reported i n thi s the s i s were to d e f i n e ovine cero id - l i po fuscino s i s in c l i n ical , pathological and genetic terms and to compare the s e fi nding s wi th tho s e of the enti t i e s i n man and dome s t ic a nimal s ..

(17) 2. REV I EW - THE NEURONAL CERO I D-L I POFUSC I NOSES. 1.. HISTORICAL A SPECTS. Some author s ( Koppang,. 1 9 7 3/74 ; Lake , 1 9 7 7 ) con s i der that the fir s t. d e s c r iption o f a neuronal cero id - l ipo fuscino s i s i n 1 826 .. (NC L ) was by Stengel. He de scribed four siblin gs , born to heal thy farmer parents. i n Norway , who showed vi sua l f a i l ure beginning at the age of 6 y e ar s , progre s s ing to blindne s s at .9 y ears.. Epi l eptic sei zure s , speech. d i f f i c ul t i e s and sen sory -mo tor regr e s s ion be came incr e a s ingly severe. There was pro found mental dul l n e s s at the age of 15 year s .. Two o f. t h e patients d ied at a g e s 2 0 a nd 2 1 year s , the other two were a l i ve aged 8 and 1 6 year s at the t i me o f h i s report .. Since Stenge l ' s report w a s a cl inical cesc ription only , the f i r s t comprehen s ive report o f NCL i s usual l y a scribed to Batten ( 19 0 3 ) , who d e s cribed "cerebral degeneration with symme trical chan ges in the ma culae " i n two s i s ters in Engl and , beginning at age s 4 and 6 years .. He noted. s l i gh t pa l lor o f the optic d i s c s , and ret inal pigmen tary chan ges with i rregu l a r poo r l y de f ined redd i sh-bl ack spo t s i n the ma culae . Batten gave the fol l owing d e s c ript ion. . . .. ·. .. .. cf. the d i sease :. " The typ i c a l featur e s a re loss o f inte l l ec tu a l. l o s s o f v i s ion , lo s s o f motor powe r . d e fe c t s seem to s tart together ,. In 1 9 14 ,. facul t ie s ,. In some c a s e s a l l three. in othe r s ment a l s ymptoms occur. firs t , visual and motor s i gn s remain long in abe yance , in sti l l o the r c a s e s the d i sease commences w i th vi sual s ymptoms , motor and men tal de f i c i t s fol low later or not at a l l .. The d i se a s e.

(18) 3. may begin any t ime during i n fancy and childhood a n d some cases pa s s rapi d l y with a fatal t e rminat ion , othe rs are slow i n the i r progr e s s . Some c a s e s show d i s tinct changes in the macul a , othe r s reti n i t i s pigme nto s a i n the pe r iphe ral r e tina , and stil l oth e r s h ave no fundus change s or do so on l y i n the l a ter stage s o f the d i sease . C l in i c a l l y there i s a great var iati on in the s ymptoms and th i s toge the r with the ir time o f appearance forms a ba s i s o f c l a s s i f i c a t ion .. Pathologi c al l y these c a s e s are e s sentia l l y the. same and the change s in the ce l l s a re s t r ikingly s imi l a r i n a l l c a s e s wh ich have come to autops y" . . . . . .. .. He omi t ted to me ntion the almo s t i nvariable occurrence o f s e i zures i n h i s patient s .. However , Batten ' s i s a l ucid description o f NCL and. Batten's d i s e a s e is a va l i d eponym .. Three En gl i sh chi l dren , produc€d. by first cous i n s , who developed fa i l ure of eye s ight at 6 years of a ge , fo l lowed by menta l deterioration, we re descr ibed by Mayou (19 04 ) .. Thi s. h a s l e d t o the eponym Batten-Mayou s yndrome .. I n 1 9 0 5 , p o s s ibly unaware o f the En g l i sh publ i c ation s , two German authors , Vogt and Sp i e l m€yer , independently de scribred a d i s e a s e o f c h i l dren which i s now con s id ered to have been a n euronal c eroi d - l ipo fu s c i no s i s and the eponymic de s i gnation Batten - Sp i e lmeyer­ Vogt syndrome i s conunonl y used . h i s d i s ea s e entity,. However , at the time , Spi e l meyer c a l l e d. "j uvenile amaurotic id iocy " a n d Vogt c a l l ed h i s. "the j uven i l e form o f amauroti c fam i l y idiocy " .. No sologi c a l con fusion had thus begun, s ince amaurotic family i d iocy was the name given to a d i sease entity of c h i l dren by Sachs.

(19) 4. ( 18 9 6 ) , 188 7 .. i n the U . S . A . ; he had e arli er reported on its o c c urrence in In En gland , Tay had de s c r ibed a s imilar d i se a s e o f ch ildren i n. 188 1 , a n d H i g i e r ( 1901). introduced the eponym Tay- Sachs d i se a s e .. The f e ature s o f Tay-Sachs d i s e a se , wh i ch ha s its hi ghe st prevalence in Ashk e na z i Jews , are weakn e s s be g i n�ing at 3 -. 6. mc�th s o f age , w ith. mental and motor deter ioration progre s s ing rap i dly a fter. 1. year of a g e .. Patients o ften e xh ibit cherry-red spots i n the macular re gion.. After. 18 month s , progre s s ive bli ndne s s , dea fne s s , convul s ion s and spa stic ity oc cur and death follows u sually arounc ( O'Br i e n ,. 3. yea r s of age. 197 3 ) .. Both Batten and Sp i elmeyer co n s idered the ir c a s e s to be d i f ferent from Tay - Sachs d i sease be cause o f d i f ference s in race pre d i spo s ition , a later age o f on set , a more protracted cour se and d i f ferences i n ophthalmolo g i c al find ings .. Vogt , e n the other hand , cons idered h i s. c a s e s to b e e s s e ntially s imilar t o tho se o f Tay and S a ch s , the only d i f ference be i n g a later a ge o f onset .. The common f in d i n gs were the. familial i n c idence , the cli n ical s i gn s o f bli n dne s s a n d i diocy , and neuropatholo g i c ally , th e d i stenti on of neuronal perikarya by lipid mate r i a l in both brain and reti n a .. Thi s characteri st i c. neuro­. patholo g i c al f inding bec ame known a s the Scha f fer-Sp i e lme yer proce s s, a fter Sch a ffer who descr ibed it i n c a se s o f Tay - Sachs d i s e a se in 1905, and Spi e lmeyer who reported it in h i s own c aseE=. in the s ame y e ar . De spite further d i f ference s betwe en Batten ' s d i sease and Tay- Sa chs d i s e a se, s uc h a s d i f ferent sta ining behaviour o f the i ntraneuronal material as well a s d i f ferenc e s in s i ze and we i ght of bra ins at a utop s y ( Zeman and Dyken, 1969}, a unitarian concept o f arr.aurotic family idiocy g a i n e d wide spr e a d acceptance..

(20) 5. The , retrospective l y , e rroneous a c c eptance o f the unitar ian conc ept ( i.e . one d i sea se proce s s due to the neuronal accumulation o f l ipid mater ia l , l e ad i ng to a va r i ety o f d i sorders , d i f fere ntiated only by age of onset). led to no solog i c a l confu s ion and a wide va r i e ty of. "l ip ido s e s" we re c l a s s i fied a s subtype s o f amaurotic fam i l y idiocy . In the l a st two decade s those neurona l l ipidoses a s so c i ated with an i nbor n error of ly so somal c atabo l i sm have been c l e a r l y d i f ferentiated . Tay-Sachs d i sease is now c l a s s i f i e d a s a lyso soma l storage di sease a c cord ing to the concept of Hers. (1965).. A f fe cted patients have a. d e f i c i en t activ i ty o f the l y sosomal en zyme hexo sami nidase A , which r e su l t s in the l y sosomal accumu l a t ion of G gangl i o s i de M2 O ' Br i e n , 1969), hence the term G. M2. Gang l io s i do s i s .. ( Okada and. The Batten­. Spi e lmeyer -Vogt syndrome o r'' j uve n i l e amaurotic fami l y i d iocy" has not y e t been de f i ned in biochemi c a l terms .. Undefined a l so , r ema in at. l e a s t two further types of amaurotic fami l y id iocy wh ich are usua l l y a l so c l a s s i fied a s neurona l ceroid-l i po fu s c i no s e s .. I n 1913, B i e l schowsky , i n Ge rmany , d e l i neated " late i n fant i l e fami l i a l amaurot ic idiocy" from both Tay- Sachs d i se a s e and j uve n i l e amau r o t i c i d iocy a s de fined b y Vog t .. He ob served severe c erebe l lar. a t rophy and cons i de red h i s c a s e s to b e identical to those o f Jan sky , who i n 1908 obs e rved four s ib l i ng s w i th a d i se a se beginning dur i ng the fou r th year o f l i fe .. A l l s u f fered from convu l s ions , lost the. abi l i ty to speak and walk and became bli n d ; d ie d between age s 4 and 6 year s .. a l l were i diotic and. I n 1925, Kuf s , i n Germany , d e sc r ibed. a d i se a s e whi c h he c a l l ed the "ad u l t form of amauroti c f am i ly idiocy" i n two s i b l ings .. The age o f o n s e t wa s i n the l a t� twen t ie s , both. patients were d emented but n e i ther wa s b l ind .. Hall evorden described.

(21) 6. a s imi l a r di seas e entity in 1938 , a nd the eponym Ku fs-Ha l l evorden di sea se is sometimes encountered in the l i terature .. A d e ta i l e d c l inical and genetic study o f 1 1 5 pa t i e n t s with j uven i l e amauro t i c idiocy from 5 0 d i f fe re n t fami l i e s in southern Sweden wa s reported by S j ogre n in 1 9 3 1 .. He found a range of onset from age. l - 19 years , with the ma j o r i ty of patients becoming b l ind at age 5 - 9 years .. He con sidered h i s ca s e s to be identical with tho se descr ibed. by Batte n , Mayou , Vogt and Spie lmeye r .. Re ferring to the l a te. i n fan t i l e form o f Ja nsky -B i e l schow sky and the adu l t form of Ku fs , he stated:. " I see no reason for con s id e r ing or di scu s s ing the se two. d i se a s e s . ". H e proposed a rece s s ive mode o f inheritanc e .. De spite Sj ogren's sta tement , a l a te i n fantile ( Ja n sky-Biel schow sky ) a n d adu l t ( Ku f s ). form a r e genera l l y c l a s s i f i ed with the j uven i l e. Mayou , Spie lmeyer , Vog t , S j o gre n ) n euro n a l c eroid - l ipofusc ino s i s . form d e s c r ibed b y H a l t i a ( 1973 ) .. e t al.. ( Batte n ,. form unde r the gene r a l heading o f To thi s l i s t can be added an i n fan t i l e. ( l 9 7 3 a , l 9 7 3b ) a nd Santavuori. et al.. The infantile form o f NCL i s character i s ed by an o n s e t o f. i l l n e s s dur i ng the f i r s t or second year o f l i fe , a very rapid cour se and b l indn e s s developing early .. Not a l l d i sease enti tie s c l a s s i f ied a s neuronal c e roidl ipo fusc inoses fit the above c a tegories exactly .. A subgroup c a l led. " ea r l y juve n i l e " was d e s c r ibed by Lake and Cavanagh ( 19 7 8 ) .. A. protracted form o f j uven i l e NCL ha s been d i f ferentia ted from the c l a s s ic a l Kuf s type by Goebe l. et al.. (197 6 ) .. An a typ i c a l juven i l e. neuro n a l c e ro i d - l ipofu sc ino si s , showing absence o f v i sual symptoms and retinal abnorma l i t i e s wa s d e sc r ibed by Greenwood and Nel son ( 19 7 8 ) ..

(22) 7. A d i se a s e e n t i ty which i s sometimes inc luded under the neuro n a l ceroid-l ipo fu scino s es i s the p i gment v a r i a n t of Ku fs d i sea se , wh ich shows a l l the features o f the l a tter as we l l a s extra -neuro n a l pigment ac cumu l a t ion ( Se i te lberger and S imma ,. 196 2 ;. Roroupian and Ro s s , 1 9 7 7;. Jerv i s a nd Pu l l arka t , 197 8 ) .. In summary , the descr iptive term "neurona l c e ro i d - l i po fusc inos i s " introduced b y Zema n a nd Dyken. ( 1 9 6 9 ) , has ga ined widespread ac ceptance .. Rea l i sa t ion tha t c e l l types o ther tha n neurons were involved i n l ipopigment storage ha s l ed t o the in troduct ion o f the term " genera l i sed c e roid-l ipo fuscino s i s ". ( Joosten. et. al. , l 9 7 3 ) .. Neu ronal or genera l i sed. ceroid-l ipofuscino s i s is synonymou s with Ba tten' s di sease and some of the amaurotic fami l y idioc ie s .. Th ese a r e umbrella terms wh ich tend. to obscure the d i f ferenc e s be twe en the var ious subtype s .. Only when. the chemical natu r e of the me tabo l ic errors invo l ved in th i s hete rogeneous group o f d i sea s e s i s e l uc idated wi l l a r a t ional nomenc l a ture become po s s ibl e .. II.. C L I N ICAL F IND INGS. The principa l c l in i c a l f indings of the huma n neuronal c eroid­ l ipo fu sc inose s , a l so c a l l ed gene ra l i sed ceroid-l ipo fu s c i n o s e s ( Jo o s te n. et. al . , l 9 7 3 ) , a re bl indne s s , l o s s o f menta l facultie s ,. loss o f motor power, s e i zure s and a var iable course i ne xorably l eading to premature death (Zeman and S i akoto s , 1973; Zeman , 197 6 ) .. There. ex i sts wide var i a t ion in age o f o n s e t , pre s ent.inq s igns and c l i n i c a l cour s e i n p a t i e n t s a f fected wj t h t-tCL .. However , a stdk ing fea ture. of the disease is tbe s i m i l adty in ase of onse t and c l in ical ccurse.

(23) 8. o f af fe cted s ibl ings for wh i ch the terms homochron i sm and homotypism are us ed re spective l y .. C l a ssi fication Various at tempts have been made to del ineate di s t i n c t subgroup s o f the neuronal cero i d- l ip fus c ino s e s , based on clinical findings , e specia l l y o n the age o f onset o f di s e a s e . d i f ferentiates l a t e i n fan t i l e. The cl a s s i cal divi s i on. (Jansky - B i e l schowsky) ,. j uven i l e. ( Batten- Sp i e lmeye r -Vo g t ) and adul t ( Ku f s ) types with re sp e ctive a ge s o f onset o f from 2 - 4 years , 4 - 8 years and 20 yea r s and o ve r ( Zeman and Siakoto s ,. 19 7 3 ) .. To thi s l i s t c a n be added. age o f onset of l - 2 years. an. i n fan t i l e type with. ( Santavuor i et al., l97 3 ; Hal t i a et al.,. l 9 7 3a , l 9 7 3b ) .. There i s by no mean s un iversal agreement on the c l a s s i f i c a t io n o f di stinct s ubgroup s a n d because o f t h e c l in i cal va r i at io n s , more recent a utho r s. ( e . g . Dekaban and Herma n , l9 74 ; Lake ,. a mul ti -di s c ip l ina ry approach .. 19 7 7 ) have adopted. Ba sed o n c l i n ical , h i s to chemic a l ,. ul t rastruc tural and e l e ctrophy s iolo g i c al f indings , Lake. ( 19 7 7 ) divi d e s. h i s c a s e s i n to i n fa n t i l e , late i n fan t i l e a n d juvenile subgroup s .. The s e. three types a n d a n adul t type wi l l be u s e d a s guide l in e s in thi s review , and Table l . I , s umma r i s e s the findings o f re cent autho r s u s i n g the c r i teria of Lake ( 19 7 7 ) .. Tho s e enti t i e s wh ich do not e xa c tl y f i t the. four subtype s a s outl i ne d in Tab l e l , I , s uch as the early juven i l e form ( Lake and C a vanagh , 19 7 8 ) , protracted juven i l e form ( Goebel et al. , 19 7 8 ) , and p i gment va rian t o f the adu l t type ( Je rv i s and Pul l arkat , 19 7 8 ) w i l l b e r e ferred to wher e approp r i a te ..

(24) TABLE l. I. Classification of the neuronal ceroid-lipofuscinoses. Subgroup. Approximate age of onset (years). l-2. Infantile. Late infantile. l. Presenting symptoms. Mental and motor retardation, visual failure. 11. EEG. White blood cell appearance. Neuronal staining. Ultrastructure. Reference. Flat by 3 yrs. Normal. PAS,SB,FQ.. Granular osmic philic deposits. Santavuori et al. (1973); Haltia et al. (l973a, l973b) et al.. 11. 11. 11. 11. Hagberg (1974). 11. 11. 11. 11. Lake (1977). 1. Mental and motor retardation. 2-5. Neurological and mental deterioration. Epileptiform. 2-4. Convulsions. Dysrhythmic with spike and wave complexes. 2. Fits, speech difficulty. Response to photic stimulation. SB,FQ.. Curvilinear or membranes in stacks. Dekaban and Herman ( 1 974). Frequent neutrophilic hypergranulation. PAS,SB,ORO FR-. Curvilinear, fingerprint, granular. Zeman and Siakotos (1973); Zeman (1976). Normal. SB,LFB,FR-. Curvilinear. Lake (1977) contd.. \.0.

(25) TABLE l. I (contd. ) Subgroup. Juvenile. Adult. Presenting symptoms. EEG. 7-10. Impaired vision. Epileptiform. 4-8. Blindness. Dysrhythmic. 6. Blindness. 31. Psychological and motor disturbance. Spikes, sharp waves, slowing. 11 and over. Myoclonic jerks, chorioathetosis. Epileptiform. 20 and over. Mental and motor disturbance. Approximate age of onset (years). PAS - Periodic Acid Schiff;. ·SB-Sudan Black;. ORO- Oil Red 0;. White blood cell appearance. Neuronal staining. Ultrastructure. SB,ORO,F£. Focal densities, Dekaban and fingerprints He rman (1974). Lymphocytic vacuolation, neutrophilic hypergranulation. LFB,PAS,SB, ORO, F£. Fingerprint, curvilinear, granular. Zeman and Siakotos (1973) ; Zeman (1976). Vacuolated lymphocytes. PAS,SB,LFB, F.Q,. Fingerprint bodies. Lake (1977). Some neutrophilic hypergranulation. PAS,LFB,F£. Osmiophilic granules. Boehme (197l). SB, F.Q,. Lipid droplets, granular. Dekaban and H e rman ( 1 974). SB,PAS10R01 F£. Granular with lipid vacuoles. Zeman and Siakotos (1973) ; Zeman (1976). LFB- Luxol Fast Blue;. Reference. et al.. Ft- Fluorescence. � 0.

(26) 11. ����� l and ophtha lmological findings Al though rare in the adul t form (Dom et al.�. 19 7 9 ) , vi sual. d i s turbance is a common and characteri stic find ing in the neurona l ceroid- l ipo fusc ino se s .. Lack o f vi sion i s commonly the presenting s i gn. in the j uveni l e form and i s con sidered highly chara cteri stic by some authors ( Zeman and Siakotos , 1 9 7 3 ; Lake , 19 7 7 ) . form Sant avuo r i e t a l .. In the i n fan t i l e. ( 19 7 3 ) repo rted that v i s ual imp a i rmen t wa s. e s tabl i shed in a l l the ir pa tients a t 18 mon th s of a ge .. In the l a te. in fan t i l e form vi sual d i s turbance usua l l y occurs later in the d i s e a s e when the p a t i e n t i s a l re ady severely debil itate d ( Zeman e t a l . �. l97 0 ) .. When bl indn e s s has e n sued in patients with NCL , the pup i l l a ry re fl e x and e l ectroret ino gram are abo l i shed .. Pat i e n t s suffering vi sua l impai rmen t may show a l l o r some o f the fol lowing ophtha l mologi cal abnorma l i t i e s: atrophy and/or waxy appearance o f the opti c d i s c , pigmentary change s of the retina and retinal atrophy .. The p i gmentary change s may be con fined•to i rregu l a r. granul a r or " s a l t a n d pepper " patterned depo s i tions i n the ma cul a o r periphery o f the r e t in a .. At o ther time s ,. "bone-spicule l ike " change s. may be seen i n the periphery o r other parts o f the r e t i n a , the so-ca l l e d retinitis pigmentosa. ( Zeman e t al.� 1 9 70 ; Zeman and S i akotos ,. In the infan t i l e type o f Santavuor i et al.. 197 3 ) .. ( 19 7 3 ) , p i gmen ta ry change. was in the form of a hypo -p i gmented dystroph i c re tinal p e riphe ry . Retinal atrophy i s a result o f degeneration and di s appe ar ance o f the l ayer of rod s and con e s and oute r nucl e a r l aye r , pe rmit t i n g vi sua l i sa t i on o f the choroid ve s s e l s . a ttenuation of retinal ve s s e l s. There i s a l so a n a ccompanying. ( Zeman et al.� l970 ) ..

(27) 12. Men tal abnorma l i t i e s The dementia o f the neuron a l ceroid-l ipo fuscinoses i s non -speci fic , but progre s s ive. ( Zeman e t aZ . �. 1970) .. In the i n fan t i l e and l a te. i n fan ti l e subgroups , men t a l dete riorat ion i s e xhi b i t e d as a retardation of normal mental deve lopment.. The children be come wi thdrawn , l o s e. intere s t in the ir e nvironment a n d stop spe ak ing o r s e eking the attention o f the i r paren t s .. Some are apathe t i c , o the r s become i rri table and. di f f i cu l t to manage .. In one to a few yea r s , the pati ents be come. comp l e t e l y un respo n s i ve to the i r environmen t ( Zeman e t aZ . � l 9 7 0; Santavuor i e t aZ.�. l9 7 3 ) .. I n the juven i l e type , the earl i e s t man i fe station o f mental d e ter iorat ion is a l o s s of a cqui red knowl e dge. ( Sj � gren , l9 3l) .. Patie n t s l o s e the abi l i ty to concentrate o r memo r i s e and show l a ck o f mo t iva t io n .. A s the d i s e a s e progre s s e s , patients usua l l y become more. apa th e ti c , but may show a ggre s s ive behaviour and rare l y psycho t i c man i fe s tation such a s parano i a a n d h a l l uc i n a t ions 197 3 ) .. ( Ze man a n d Si akoto s ,. Dementia may be present i n the adul t form , but i f e n countered. is l e s s s evere than i n the other forms of NCL and doe s not affect a l l men t a l facul t i e s .. Fine e t aZ .. ( 1960 ) , i n a review o f the l i terature. r e corded tha t mental d i sturban ces occurred i n n e a r l y a l l 18 case s , but the i r own patient rema i ned mentally a l ert .. Motor s ymptoms In the i n fan t i l e subgroup , motor development c e a s e s a round 18 months of age .. Late r , a ffected patie n t s show gene r a l i s e d hypoto n i a ,. trun c a l and l imb ataxi a , e xa ggerated tendon refl e xe s and c lonu s .. At a. s t i l l l ater s ta ge patients show hypotoni a and epi sodes o f opi sthotonus.

(28) 13. w i th hype rton i c flexion in the arms. p.nd e xtens ion o f the l e gs .. I n the. te rminal stage there is pe rmanent flexor tonus in a l l l imbs ( San tavuo ri et al.� 19 7 3 ) .. Patients w i th the l ate infan t i l e and juven i l e fo rms o f NCL develop motor d i s turban ces varyin g from an i n i t i a l pa rtial l o s s o f ab i l i ty to per form coordinated movements to a terminal incapa c i t a t i ng r i gi d i ty with f l e xion contracture s o f n e ck , Often an i n i t i a l clums i n e s s develops with ataxi a o f the l e gs .. trunk and e xtremiti e s .. i nto a n in coord i n ation o f ga i t. Pat ients may show di fficulty i n i n i t i a t i n g. movement , but s u c c e e d i n c arrying o u t a movement o n c e s tarte d .. The. uppe r extrem i t i e s re tain the i r use longe r , d e sp i te abnormal po stur ing and hype rk ine s i a .. Mastication and gluti t io n are impeded and the re i s. i mp e r fect articulation o f speech ( Zeman and Dyken , 19691.. Motor di s turban ces in the adult form a re usua l l y l e s s i n c apacitati n g .. A f fected patients may s u f f e r from cerebe l l ar atax i a. and show sti f fn e s s , pecul i a r po sture , l o s s o f associated moveme n t s and ak i n e s i a ( Zeman et al.�. 1970).. Se i zure d i so rde r s and e l ectro-encepha l ogram ( EEG). findings. Convul s i ve epi sode s a re highly cha r a c ter i stic of the late i n fant i l e type of neuronal ceroid-l ipo fus c inosis and commonl y occur i n the i n fant i l e , juve n i l e and adult type s Santavuor i et al.� 1973;. ( Zeman and Dyk e n , 1969;. Dekaban and Herman , 1974;. Dom et al.� 1979).. S e i zure s , which are often the presenti n g s i gn in the juve n i l e form , a re most conunonly o f the grand rrr1l typ e , but may take the form o f myocl onic or petit mal spells or may occur a s tonic spa sms ,.

(29) 14. psychomotor, ma s s ive myoclonic, akinetic and cerebe l l ar f i t s .. In the. t e rminal pha se , regardl e s s of a ge , patients become comatose with a myoc l on i c hyperexci tabi l i ty .. The frequency o f convul sion s vari e s. from a few t ime s dur ing a y e ar to a vi rtu al status epilepticus�. and. when presen t , convul s ions tend to increase i n frequency towards the end of the course of the d i se a s e. ( Zeman et al . �. 19 7 0 ) .. The EEG abnorma l i t i e s con s i st o f dys rhythmia in add i t ion to sp ike and wave complexe s and s catte red h i gh amp l i tude de l t a wave s . S e r i a l EEG ' s re flect the progre s s ive worsen ing of c l in ical s i gn s and s ymp toms i n the form of deteriorating background rhythm and l e s s p ronounced p aroxysmal a c t i v i t y t h e E E G become s i soelectr i c .. ( Zeman and S i akoto s , 1 9 7 3 ) . Zeman et a l .. ( 19 7 0 ). U l t imat e l y. reported that. pho t i c st imu l a t ion evok ed occipi t a l sp ike s in some of the i r pat i e n t s s u f fe r i n g from the l ate i n f ant i l e a n d j uven i l e forms o f NCL . r e spon s e s coul d be reco rded in b l ind patient s .. Th ere wa s no re�ponse. to pho t i c s t imu l ation i n the p at ients of S an tavuor i et al . Dom e t a l .. No. ( 19 7 3 ) .. ( 19 7 9 ) reported a nrrl d d i f fuse dysrhythnUa a s the i r EEG. findings in two patients s u f fe ring from the adul t form of NCL .. Rad i 0 o gic.<'ll f indings _ _ _ _ X-ra y s and pneumoencephalography may reve a l a total incre a s e in t h ickn e s s of the calvaria and varying degr e e s o f c e rebral and c e rebe l l ar a trophy, enl arged subara chno i d space s and d i l at ion of t he ven t ri c le s .. In gene ral th e d e gree o f cerebral and c e rebe l l ar a t rophy. i s more severe in ent i t i e s w i th early onset ( Hagbe r g et a l . � Zernan e t a l . �. 1970 ;. Fine e t a l . ,. 1960 ) .. 19 7 4 ;. Patients w i th the in fant i l e. a n d l a t e i n fa n t i l e forms o f NCL a l so show microcephal y ( Zeman e t a l . � 1 9 7 0 ; Hagber g e t al . ,. 19 74} ..

(30) 15. Le ukocyte abnorma l i t i e s Lymphocytic vacuolation w a s fi r s t obse rved by Bagh and Hort l i n g (1948) in j uveni l e neuronal cero id-l ipo fus cino si s .. Th is obse rvation. has been con fi rmed by numerous autho r s ( e . g . Rayne r , 1962; Zeman and S t routh , 1967; Schwen demann 1977;. ,. 1976;. S chuurman s Stekhoven et al.�. Noonan et al.� 1978; Nakano et al.� 1979).. vacuo l a ted lymphocyte s va r i e s from 0 . 5. -. i n indi vidual c a s e s at di f fe rent time s .. The percentage o f. 77% with a high fluctuation. Vacuolation i s not present. i n the i n fantile form ( Santavuori et al.� 197 3; Lake , 1977). Al though some autho rs. ( e . g . Schuurman s Stekhoven et al.� �976; Lak e ,. 1977) cons ider the absence o f vacuo lated l ympho cytes a characte r i stic. finding. in. late. in fan t i l e NCL , others. ( W i t z l eben , 1972; Zeman and. S i ako to s , 197 3; Schwendemann , 19761 repo rt that l ympho cytic vacuo l a t ion may be present in th i s subgroup .. Vacuol ated l ymphocytes have not. been reported in the adul t form of NCL . S chuurmans Stekhoven. et al.. Schwendemann. (1976) and. (1977) empha s i ze that vacuola tion o f. l ymphocytes i s not spe c i f i c for the neuronal ceroid- l ipo fus cino s e s .. Az urophi l ic hype rgranul ation o f neutrophi l s has at one t ime o r another been reported in a l l forms o f NCL ( S antavuor i et al.� 1973; Zeman and Dyken , 1969; Boehme et al.� 1971).. Both l ympho cytic. vacuo l a t ion and n eutrophi l i c hypergranul a t io n may be found in parents a n d s ib l ings of a ffected pat ients wh ich show the se abnormalities , thus s ugge s t ing that the l e ukocyte abnorma l i t i e s behave as marke r s for the mutant gene ( Rayn e r , 1962; Zeman and Dyken , 1969)..

(31) 16. III .. PATHOLOGICAL FINDINGS. Al though neuronal cero i d - l ipo fusc i no s i s man i fe s t s a s a ne rvous d i sorder , vi sceral invo lvement i s wide spre a d , and some authors ( e . g . Joosten e t al.3. 19 7 3 ; An z i l e t al.,. 1 9 7 5; Schwendemann , 1 9 7 6 ). pre fer the term gene r a l i s e d cero i d - l ipo fus cino s is .. It appears that. on l y in neuron s , the forma t ion and accumul a t ion of auto f l uore scent l ipopi gme n t s l eads to di sturbance of func tion with character i st i c c l inical man i fe s tation s .. Gro s s f indings Gro s s abnormal ities d ue to NCL are re stricted to the skul l , brain and spi n a l cord and tend to be more severe in e n t i t i e s w i th early onse t .. In the i n fant i l e form , Hal t i a et al. ( 19 74 ) , Sant avuori et al.. ( 197 3a ) , Hagber g et al.. ( 1 9 7 4 ) , reporte d pronoun ced thickening o f. the sku l l bone s , with a thick layer o f g e l a tinous t i s sue on the inner a sp e c t of the dura mater and thickening and oe dema of the l eptomen inge s .. The brains were noted to be smal l , due to d i ffuse. cerebral gyral a trophy .. Brain we i gh t varied from 305 - 4 2 0 g .. The. cerebral cortex was 1 . 0 - 1 . 5 mm thick , yel lowi sh grey and g e l a tinous in appearanc e .. The ventri c l e s were d i l ated , whi l e b a s a l gangl i a and. tha l amus we re shrunken . a trophic fo l i a .. The cerebe l l um wa s sma l l and firm w i th. The spinal cord w a s l e s s a f fecte d , but the grey. matter i n the ante r ior horns was conspi cuously yel l ow .. Wi t h the. e xception o f a t roph i c opti c n e rve s , there was no g ro s s abnormal ity of cran i a l n e rve s , spinal roots and s c i at i c nerve s ..

(32) 17. Zeman e t al.. ( 1 9 7 0 ) , in the i r review of the neurona l c e ro i d -. l ipo fus c i no se s , commented that the mos t s t r iking gro s s abnorma l i t i e s wer e ce rebra l a trophy and r educed b r a i n wejght . we i ghts f o r the l ate i n fanti l e form w e r e 2 5 4 j uven i l e fo rm 4 10 - 1 2 0 0 g .. -. The range s o f b r a i n 8 0 0 g , a n d for the. Coronal s e c t ions of brain reve a l e d a. cha racte r i s t i c yel low-brown colour o f the cortex and to a l e s se r e xtent , o f t h e subco r t i c a l grey ma t te r .. Th icken ing o f the skul l is. usua l l y present in both type s , wh i l e opt i c ne rve atrophy and ce rebe l l a r atrophy are present in the l ate i n fan ti l e , but need not be pre sent in the j uvenile fo rm .. The adult form o f NCL shows cerebe l l a r at rophy , with gap in g fo l i a a n d a f i rm reduce d c e n t r a l whi t e ma tter a t autopsy ( F i n e e t a l . , 1960; Boehrne et al. �. 1971) .. I n a recent case Darn e t al.. ( 19 7 9 ). 1. reported a gene r a l i sed cor t i c a l a trophy of ce rebrum and ce rebe l l um a n d a mi l d d i l at ion o f the vent r i c u l a r sys tem.. Hi s topa thology CNS and retina :. The characte r i s t i c mic ro scopic chan ge in ne rve c e l l s. o f brai n and retina was f i rst de s c r ibed by Sp ie l meye r. ( 19 05 ) .. He. noted an irregu l ar d i s tent ion o f n e uron s due to the intracytop l a smic depo s i t ion of a yel low coloured granul a r materia l . now con s ide red to con s i st o f the. l ipopi grne n t s. ( Zernan and Alper t , 1963; S i akoto s e t a l . � predominating i n the l ate i nfanti l e ( Braak a n d Goebel , 197 8 ) forms .. ,. Thi s mate r i a l i s. ceroid dnd lipofuscin. 197 0 ) , w i th ceroid. (Wo l fe et a l . �. 197 7 ) and j uven i l e. Accumul a t ion o f l ipopigrnents i n. ne rve c e l l s i s the common morpho lo g i c feature of a l l forms o f NCL . Howeve r the e xtent o f neuronal involveme n t and sequelae o f p i gment.

(33) 18. ac cumu l a t ion such as neuronal at rophy and death , as we l l as neurogl i a l and me sode rmal invo l vement va r i e s between the di fferent type s , a lbe i t wi th con s i derable ove rlap .. The in fant i l e form shows a qua l i ta t i ve ly ste reotyped h i stologi cal p i c ture with the only va r i a tion be i n g the de gree o f t i ssue a l terat ion , according to Hal t i a et al.. ( 19 7 3a , 1 9 7 3b ) .. They stud i e d the. mi croscop i c pathology using bra i n b iop sy material in twe l ve c a s e s and autopsy mate r i a l in three c a s e s .. In t he ce rebral cortex the. cytoarchi tec ture was seve r e l y d i sturbed , w i th marked loss of neuron s . The rema i n i n g neuro n s had l arge ve s i c u l a r nuc l e i and the cytop l a sm was d i stended by granular mate r i a l or i rr e gular homogeneous i n c l u s i on s . Coa rse granule cont a i n ing macropha ges , o ften wi th two or thre e nuc l e i were seen be tween the cort i ca l neuron s .. I n the biopsy mate r i a l ,. neuronophagia was not a p romi nent feature , where a s i t was common i n the autopsy ma te r i a l . astrocyto s i s ;. There wa s intense cortical fibr i l l a ry. the a s t rocyte s had coa r s e proce s s e s and con ta ined. granular s torage mate r i a l s imi l a r to that o f the neuron s .. Other. change s were a to tal l o s s of neuron s from the cerebe l l ar cor te x , w i th hyp e rtrophy o f g l i a l c el l s , which c on tained con s i derable amounts of s torage mater ia l .. Neurons i n the bra in stem and basal gangl i a. were swo l l e n by l arge amoun t s o f char ac te r i s t i c s torage mat e ri a l . The whi t e ma tter showed hypertrophy and an incre a s e i n numbe r o f a s trocyt e s with c ha ra c te r i s t i c g r anul a r i nclusion s .. In o l de r patients. there was loss of a xons and mye l in she a th s with a n accomp anying f ibri l la r y a s trocyto s i s .. The c ha racter i s tic in c l usions were auto­. fluore s ce n t in u l travi o l e t l i ght , we re a c i d- fast , s t rongly PAS­ posi tive and stained inten s e l y b l ack wi th Sudan b l ack B..

(34) 19. The retina showed severe a trophy , w i t h complete l o s s o f gangl ion cel l s, rods and cone s and bipo l a r ce l l s , prol i feration of g l i a l e l emen t s and l a rge p igment l a den macrophage s .. No mye l i n sheath s were l e ft i n. the atroph i c and g l i o se d opt i c n e rve s.. The l a te i n fan t i l e form showed fine and coarse granul a r mate r i a l wi thin the ma j or i ty o f neuronal per i ka rya , which we re mi l d l y d i st ended, according to a recent d e s c rjpt ion o f l i gh t microscop i c find ings by de Baecque e t a l .. ( 197 6 ) .. The c e rebe l l um s howed a reduct ion i n the. number of Purk i n j e c e l l s , a strik ing l o s s of granul ar ce l l s and a prominent Bergrnann c e l l gl io s i s .. Neuron s i n the spi n a l cord were. e xtreme l y d i s tended with frequent d i spl a cement of the nuc l eu s and Ni s s l sub s tance .. The granular ma t e r i a l showed a ye l l owi sh fluore s c ence. under ul travi o l e t l i·]ht .. Sudan black and PAS po s i t ive mate r i a l was a l so. found i n the cytop l a sm o f g l i a l and e ndothe l i a l cel l s .. The whi te matter. change con s i s te d o f mi l d demye l in a t ion and pro l i feration of a s t rocytes. Se i te lberger e t a l .. ( 19 6 7 ) de s c r ibed a s econd type of cytopl a smi c. i n c l us ion i n n erve c e l l s , these being i r r egularly shaped homogen eou s spheroidal bod i e s o f di�ferent s i ze s , whi ch occur i n a dd i t ion to the granular depo s i t s .. These bod i e s were encoun tered mo s t regu l a r l y i n. the substantia n�gra but occurred e l s ewhere in the bra in.. The retinal l e s ions cons i s t o f l o s s of rods and cone s , var i ab l e ac c umulation o f l ipop i gment i n ganglion c e l l s , and a var i ab l e degree o f opt i c nerve a trophy ( Zeman , 1 9 7 6 ) .. The j uven i l e form i s charac t e r i s e d by moderate d i stent ion o f.

(35) 20. n e uronal per ikarya due to the ac cumulation of auto f l uo re s cent p i gments . Ne rve ce l l l o s s i s usua l ly not very marke d , a l though the ce l l s o f the c e r ebe l l a r g ranul a r. l ayer may be mi s s i n g or reduced in numbe r .. P i gment. a l so ac cumu l a te s i n a s trocyte s wh ich usua l ly show mode rate p ro l i ferati on . The whi te ma tter shows a d i screte a s trocytic g l ios i s .. In the r e t i n a ,. the gangl ion c e l l s conta in con s i de r able amount s o f l ipopi gmen t .. The re. is complete l o s s of rod s and cones and outer nuclear l ayer ( Ze ma n , 197 6 ) . Wh i l e agre e i n g tha t ne uron a l l o s s i n the j uvenile form i s mod e ra t e , B raak and Go ebe l. ( 197 8 ). report a n almost complete to total l o s s o f. s ma l l p i gment - l aden ste l l ate cel l s i n the i so - cortex o f a n 1 8 y e a r s o l d patien t .. They postulated that the s e l ective i nvolvemen t o f the se. local c i rcui t n e urons may be causal l y re l a ted to the fun c t i on a l impai rment o f the b ra in .. The adult fo rm shows ub iqui tous ac cumulation of granul a r l ipo­ p i gment in neuron s .. There i s l o s s o f Purk in j e cel l s and granul a r. c e l l s i n the c e rebe l l um .. Sph e roidal i n c l usions may b e s e e n i n t h a l ami c. n e urons an d i n the substantia ni gra ( Boehme e t al . , 197 1 ) . abnorma l i t i e s had not been d e s c r ibed ( Zeman , case o f Dam e t a l .. ( 19 7 9 ) .. Re t i n al. 197 6 ) unt i l the recent. In the r e t i n a o f the i r p a t i e n t they found. the e xternal s e gme nt o f mo s t rod ce l l s to be d i stended w i th brown­ y e l low granule s .. The r e was a l so r e t i n a l a trophy and l o s s o f gan g l io n. c e l l s w i th b a l l oo n i n g o f per s i stent neurons b y ce roid-lipoLu��iu granule s .. Seve r a l autho r s Ro s s , 1977 ;. Je rvi s. ( Se i t e l be rge r and S imma , 196 2 ; Horoup i an a n d a n d Pul l a rkat , 197 8 ) have de s c ri b e d a p i gme n t. variant o f Ku f s d i s e a s e o r adult n e urona l ceroid- l i po fu s c i no s i s ..

(36) 21. Thi s d i s e a se r e s embl e s Ku fs d i s e a se c l inical l y , but patho l ogically pre sents add i t ional featur e s in the form of extra -neuron a l p i gmen t depo s i t s whic h are mo s t obvious in t h e globus pa l lidus a n d subs tantia nigra .. The nature o f th i s extra-neuronal p i gment and. i t s r e l at ion to ceroid and l ipo fusc in remain unknown. ( Je rv i s and. Pul l a rkat , 1 9 7 8 ) .. V i s c e r a l l e s ion s :. A systematic exami nation o f v i s c e r a l i nvo l vement. in three c a s e s of the j uve n i l e form of NCL us ing l i gh t and ul travio l e t micro scopy wa s repor ted by Kr i s ten s son et a l.. ( 19 6 5 ) .. They. found depo s i t s w i th staining and fluore scence char acteri s t i c s simi l a r to tho se in neurons o f the central ne rvous sy stem in c e l l s o f the fo l lowing t i s su e s or organ s :. dors a l root gangl ion c e l l s , autonomic. nerve c e l l s of the intestinal wal l , perinucl ear re gion o f myocard i a l mus c l e c e l l s , epithe l ium o f the d i s t a l part o f col l e c ting tubul e s i n the k idneys , l a rge reticulo-endo the l ia l cel l s i n spl e en, thymus, lymph nod e s and bone marrow , parenchyma l and Kup f f e r c e l l s in the l iver .. Variable amounts of f l uorescent granular mate r i a l we re a l so. found in the anterior lobe thyro i d s and gonad s .. of the pituitary, pancr e a s, adrena l s ,. Inc l us ions in the thyroid gl and s we re also. d e s c r i bed by Dayan and Tri ckey ( 1 9 7 0 ) .. Mi l ey et a l .. ( 19 7 8 ). de sc ribed the pre sence o f s e a -b l ue h i s t iocyte s i n the bone marrow of a 6 yea�s o l d boy with j uven i l e NCL .. U s i n g the Sudan IV s t a i n i n g te chn ique Dolman and Chang ( 1 9 7 2 ) e xamined f ro z e n and paraf fi n embedded s ections o f a l a rge number o f v i s c e r a l organs i n two c a s e s o f l a t e in fanti l e NCL .. They found.

(37) :' 2. i n t racytopl q smic Sudan. IV. po s i t ive staining ma ter i a l , varying from. s i n gle sma l l i n c l us ions to mul tip l e granu l e s in c e l l s of hea r t , aorta , l un g , pituitary , thyro i d and adren a l g l and s , bone marrow , l ymph nodes , Nowhere wa s there. k i dne y , urinary bladde r , striated mu s c l e and skin .. abno rmal d i s t e nt ion o f the ce l l s as o ccurs in the n e rvous Simi l ar findings were repo rted by de Bae cque et a l .. V i s c e r a l i nvo l veme n t in the in fant i l e type by Hal ti a et a l .. ( 19 7 3b ) .. system.. ( 19 76 ) .. o f NCL was d e s c r ibed. They found abundant cytop l a smi c depo s i ts. i n epi the l i a l c e l l s o f the thyro i d , panc re a s , kidney and t e s t i s , whi l e hepa tocytes seemed not to be invo l ve d .. Characte r i s t i c. i n c l us ions we re al so found i n ske l e tal a n d cardiac mu sc l e , a n d smooth mus c l e o f the inte s tinal wa l l .. Large macropha ges with sudanoph i l i c. granu l e s i n the i r cytop l asm were seen i n sp l e en , l ymph node s , bone ma r row and lamina propria of inte s t inal mus co sa.. A lveo l i and. interstitial t i s s ue o f the l un g and Kup f f e r cel l s in the l iver a l so contained a l ar ge number of granul e s , whi l e fibrocyte s se emed to be f r e e o f the mat e r i a l .. Mo s t repor t s o n the adu l t form o f NCL fail to men t ion v i s ce r a l invol vement , which could p ar ty b e due to i n complete e xamin at ion , a l though F ine et a l .. ( 1960 1 mention negat i ve findings.. Kornfe l d. ( 19 7 2 ) repo r t s invol vement o f Kup ffer c e l l s , glomerular podocyt e s a n d l a rge mononuc l e a r s i n spl een , bone marrow and lung.. Dom et a l .. · ( 19 7 9 ) showed invo lvement o f l iver , heart and ske l e t a l mus c l e . : I n the p i gmen t variant o f Kufs d i sease , Je rvi s and Pul l a rkat ( 19 7 8 ) d e s cribed the presence o f l ipo fuscin i n l iver , spl ee n and kidney ..

(38) 23. Ultrastructural finding& The e l e ctron microscope has be en a us e ful too l i n d e l i neating the neuronal cero i d - l ipo fusc inoses from the gangl iosido se s .. Te rry and. Korey ( 19 6 0 } d e sc ribed the ul tra structure o f the neuronal i n clusions The fi rst ultrastructural observations of. in Tay-Sachs d i s e a se .. the i n c l u s ions of j uven i l e amaurotic idiocy were repo rted by Zeman and Donahue. ( 19 6 3 ) .. The autho r s used the te rm " j uve n i l e " to. d i s t in gui sh the i r cases from Tay- Sachs d i s ease , however i f the guide l i n e s of Table. 1. I. a re used the i r patients woul d probabl y be. cl a s s i fi e d w i th the l ate i n fa n t i l e subgroup of NCL .. The y. ob served. pl eomorphic memb rane bound l ipop i gmen t bo d i e s o f va r i ab l e s i ze in neuro n a l p e r i k a rya , glia and e ndothe l i a l c e l l s .. They de scribed two. cha racte r i stic i n ternal structure s, i . e . the l a rger bod i e s d i splayed irre gul a r aggre gations of merrb ranous ma terial which fo rmed round , oval or tubu l a r s tructures whi ch gave the c ro s s - se c t i o n a mul t i locular appe a r a nc e , whi l e the smal l e r bod i e s conta ined den s e granul ar mat e r i a l i n wh ich tubu l a r structur e s w e r e commonly seen .. Koe n i g e t a Z .. ( 1964 ). demons trated a c id phosphata se a c t ivi ty in i n c lusions o f NCL , thus ind i c a t i n g the i r probab l e lyso somal location . .. The mul t i locular bod i e s are a l so d e s c r ibed as mul t i - l ame l l ar cyto some s 1968 ) .. (Gonata s e t a Z . , 1 9 68 ) , and curvi l i near bod i e s. (Duffy et a Z . ,. The l atter descr ibed the bo d i e s a s be i n g i rr e gu l a r i n outl ine. and shape and having a r an ge in size from 0. 2 - 2. 6. �m.. The bodie s. wer e compo s e d o f short curvi l i near pro fi l e s wh ich were e i ther mul t i ­ d ir e c t io n a l o r r a n a s hort para l l e l cour se presenting a tubular appe a r an c e .. A few pro f i l e s formed comp l ete c ir c l e s a n d a t h i gh. magn i f i ca tion the pro f i l e s were i n part compo sed o f f i n e o smiophil ic.

(39) 24. granul e s .. At time s the curvi l i near body was l i ned by a l imi t i n g. me mbrane b u t more o ften the e xternal bo rde r was formed b y c urvi l inear pro fi l e s s imi lar to tho se seen throughout the ma s s . i n c l u s i o n body de s c ribed by Seitelbe rger et al .. The sphe ro idal. ( 19 6 7 ) wa s shown to. con s i st o f fine , highly el ectron den se granul e s among s t wh ich curvi l i n e a r pro f i l e s cou l d be d i s c e rned .. Another st ructure rare l y. s e e n w a s compo sed o f moderat e ly broad d e n s e para l l e l l i nes e xtending a c ross the i n s ide o f an ovoid body bounded by a membrane .. I n c l u s ions f ro m 1 . 0 - 4 . 0 ).liD in dia.f.1eter with a fingerp rint-l ike pattern , con s i sting of a serie s of c l o s e l y packed paired de nse membran e s , e a ch p a i r separated from the next by a space , were des cribed by Suzuk i e t al.. ( 19 6 8 ). in the j uve n i l e form o f NCL .. " Each dense l ine. me asured about 2 7 � in thi ckne s s , the c l ea r zone within the pairs mea sured 14 to 2 0� and the space between the pairs was 2 5� wide . " a l so found inclusions 2 . 0 - 5 . 0. ).l iD. They. in diame t e r which re sembled the. memb ranove s i cular bodie s first described by Gonata s et al.. ( 196 3 ) .. The s e bo d i e s "were compo sed o f i r regularly fo lded wavy membranes 6 5 t o lOO� th ick and o f s l i ght l y e l ectron dense ve s i cul a r structure s . " I n c l u s ions showing both f ingerprint pattern and membranove s i cu l a r f e a t u r e s were occa s iona l ly observe d .. Yet a no ther type o f body pre sent. i n the n euronal perikary a was mode rately dense , granular and lysoso me ­ A t t ime s the s e bodies conta ined fine , curve d or straight l in e a r. l ike .. s tructure s . Me mbrane bound l ip i d bodies con s i sting o f a d e n s e granular matrix which parti a l ly s urrounds l arge empty space s were d e s c r ibed by Zeman et al .. (1970 ) .. They cons i dered the s e to be very s imi lar to the. l ipo fus ci n granule s found in senescent brains ..

(40) 25. The res idual bodies o f the adul t form o f NCL usua l l y contain granul a r ( Ko rn fe l d , 1 9 7 2 ) and o ccasionally additional membranous components ( Chou and Thompson , 1 9 7 0 ) .. Dom et al.. ( 19 7 9 ). found two Some. typ e s o f neurona l incl usion s i n the i r case o f Ku fs d i s e a s e . appeared l ik e typical l ipofusc in , but the ma j o r ity conta ined curvi l inear pro fi l e s .. In the in fant i l e form , the o s mioph i l i c s torage. mat e r i a l i s compo sed o f globular p a rt i c l e s 0 . 2 - 0 . 5 �m in d i ame ter and of l a rger aggre gate s up to 3 . 0 �m in di ame te r .. Both types o f. i n c l u s i o n s a re s urrounded by a un i t membrane and have a n e l e ctron den se homo geneous and fine l y granul a r internal s t ruc ture .. Occ a s ion al ly ,. a l ame l l a r o r membranous s t ructure may be s e e n within the granul ar mat r i x ( Ha l t i a et al ., 1 9 7 3b ) .. Gonatas et al.. ( 196 8 ) , and Du f fy et a l.. ( 19 6 8 ). attempted to. co r r e l a te u l t r a s t ructural findings wi th c l in i c al l y d i f fe rentiab l e e n t i t i e s o f t h e neuronal ceroi d - l ipo fus cino s e s . et al .. Howeve r , Towfi ghi. ( 19 7 3 ) , us ing the material o f Gona t a s , came to the con clus ion. that the pl eomorph i sm of the reported inclus ion s i n l ate i n f an t i l e a n d j uven i l e amaurotic fami l y idiocy probab l y re fl e c te d man i fes tations of a s ingle o r closely rel ated d i sorder s .. They a l so con fi rmed that. the n ature o f the various i n c l us ions did not change i n t ime .. Both. the s e observat ions had a l r e ady been s tr e s s e d by Zeman et a l .. ( 19 7 0 ) ,. who pointed out that i n gene ral the curvi l inear type o f i n c l us ion p redomi n ate s i n the l ate in fanti l e form, whi l e the finge rprint patterned i n c lu s ions a r e more common i n the j uven i l e form of NCL . obs e rvation wa s conf i rmed b y Goebel e t a l .. Th i s. ( 19 7 9 ) who s t ate d :. "Although the c l a s s ical subgroups o f NCL contain e l e ctronmi c ro s cop­ i c a l l y we l l de fined res idual bodi e s , permitting d i s t i nction o f the.

(41) 26. l ate in fant i l e type from the j uven i l e type , the u l tra s tructural di fferences a re more of a quant itat ive tha n of a qua l i tative nature . ". Numerous autho rs have con f i rmed v i s ce ra l involvement ele c tron ­ mi croscop i c a l l y i n virtua l ly eve ry organ e xamined i n the i n fant i l e ( Ha l t i a e t al . , l 9 7 3b ) , l ate i n f an t i l e. ( e . g . Du f fy e t al., 1968 ;. Andrews et al . , 19 7 1 ; Dolman and Chang, 1 9 7 2 ; de Bae cque et al . , 1 9 7 6 ) and j uve n i l e. ( e . g . Carpenter et al ., 19 7 2 ; Tow fi gh i et al . , 1 9 7 3 ;. Mi l e y e t a l ., 1 9 7 8 ). forms o f neuronal cero i d - 1 ipo fus c i nos i s .. E l e c t romi cro s cop i c a l ly , vi sce ra l i nvo l ve ment in the adult from has bee n reported by Korn fe ld ( 19 7 2 ) and Dom et a l .. i. ( 19 7 9 ) .. Of d i a gno s t i c s i gn i fi cance i s the o ccurrence o f re s i dual bo d i e s i n s k i n ( Do l man e t al . , 1 9 7 5 ; Ceute r i ck e t a l . , 19 7 6 ; Farre l l and Sumi ,. 1 9 7 7 ; S ipe and O ' b rien , 19 7 9 ) , sural n e rve ( Joosten et a l . ,. 1 9 7 3 ) , autonomic gangl i a and mac rophage s i n rectal mucosa ( Lake , 1 9 7 7 ; Lake and Cavan agh ,. 19 7 8 ) and appendix (Van H a e l s t and Gabree l s , 1 9 7 2 ;. Rap o l a and H a l t i a , 19 7 3 ) , l ympho cyte s Stekhoven et al . , 1 9 7 6 , 19 7 7 ;. ( Schwendemann ,. 1 9 7 6 ; Schuurman s. Noonan e t a l . , 19 7 8 ; Baumann and. Marke sbe r ry , 1 9 7 8 ; Nakano e t al . , 1979 ) , u r i n ary sed iment ( de Ba e c que , 1 9 7 5 ; Arrnstrong e t al . , 19 7 7 ) and ske l e t a l muscle ( Goebel e t a l . , 19 7 5 ; Dom e t a l . , 1979 ) .. IV.. B IOCHEMICAL F INDINGS. The l ipopigments The pigment found in nerve and muscle cel l s of aging mammal i an. individuals and conunonly referred to as "wear and tear" pigment , was termed " l ipofuscin " by Borst in 19 2 4 .. A lipofuscin-l ike pigment.

(42) 27. wa s obse rved i n l ive rs o f rats with d i eta ry c i rrhos i s by Li l l ie et a l . , i n 194 1 and gi ven the name " ce ro i d " .. The d i s t i n ct ion betwe en l ipo fuscin and c e roi d , based on hi sto­ chemi c a l and ul tras tructural ground s , was not clear unt i l S i ak otos et a Z. ( 19 7 0 ). human brain .. i sol ated two d i s t i n c t l ipop igment fractions from. Both fract ions cou ld be i so l ated from brains o f pat i e n t s. wi th any type o f neuron a l cero i d - l ip fuscino s i s .. Howeve r , one fraction. was o r i g i na l l y i sol ated from b r a in s o f aged but othe rwi se heal thy i n d i vidua l s and was therefore terme d " l ipo fuscin " .. The o th e r. pigment fra c t io n was given the n ame " ceroi d " a s i t was f i r s t obt a ined from the bra i n o f a pati ent d i a gno sed as suffering from cero i d s to rage d i s e a se. ( Levine e t a z . ,. 196 8 ) .. The p i gmen t s could be. d i f fe re n t i ated on the ba s i s o f ultrastructure , ma s s and ult ravio l e t f l uore scenc e .. Both cero i d and l ipo fus cin conta ined l y so somal. hydro l a s e s , thus reve a l i n g the i r n a ture as re si dual bod i e s. ( Koenig. e t a Z . , 196 4 ) .. Furth e r s tud i e s ( S i akotos et a l . , 1 9 7 2 , 19 7 3 ). showed that. pur i f i e d l ipofuscin is a b l a ck -brown p i gment with a d e n s i ty of 1 . 0 1 . 0 5 and i s s t able i n s a l t sol ution s , upon che l a t i on and ion exchange . I t h a s a c a t i on compo s i t ion o f 2 4 0 ppm o f Ca , 300 ppm o f Fe and 4 0 0 ppm o f Zn .. TWo-dimen s ional thin - l ayer chromatography reve a l s mo s t. o f t h e we l l d e fined pho spho l ipids , whi le the major p a r t o f the s o l ubl e l ipop i gment phas e rema i n s at the place o f appl ication and c on s i s t s of l ipid polyme r s , wh ich are 9 2 % neutral and 8 % a c i dic ( Zeman , 1976 ) .. Taubol d et a l .. ( 19 7 5 ) i n the ir s t udy o f the. chemi c a l n a t ure of l ipo fus c i n i so l ated from normal human brain ,.

(43) 28. found the mo lecular we ight o f pur i f ied l ipofu scin to be between 6000 - 7 0 0 0 d a l tons .. I n fra-red , u l trav i o l e t , neutron magnet i c. re sonance a nd fluorome tric spectra i n d i c a ted the predominantly l ipid nature of l ipofu s c i n .. They sugge s ted that l ipofusc i n con s i s t s. ma inly of polyme r i c l ipid and phosphol ip id structure s a l ong with amino acids bound to the l ipids or a s i nc l uded pro te in s .. Ceroid i n the pu r i f ied state i s a ye l low pigment with a d e n s i ty of 1 . 25 - 1 . 30 .. It occurs i n b r a i n s o f patients wi th NCL , l ive r s. from rats w i th d i etary c i rrhos i s and i s the age pigment i n Drosophi lae . I n cont ra s t to l ipofu sc in , c e roid i s un stable in sa l t so lutio n , upon chel ation , and ion e xchange ;. i t s c a t i o n composition i s 960 ppm o f. ea , 1 5 0 0 ppm o f Fe and 1 10 ppm o f zn .. Two -d imen s io na l thin-l ayer. chroma tography shows normal sphingol ipid s .. Compa red wi th l ipo fusc i n. a sma l l e r amount o f the so lub l e l ipopigment pha se i s re ta ined a t the pl ace of app l ica t io n , which in thi s c a s e con s ists o f 5 7 % neutral and 4 3 % acidic l ipid po l yme r s ( Zema n , 1 9 7 6 ) .. The u l tras tructure o f the two c l a s se s o f l ipop igment i s no longer con s idered a u s e ful ind i c a t ion o f l ipopigment spec i e s , e xcept for the chara c t e r i s t i c "curvi l in e a r c eroid " found in NCL ( S i akoto s et al. ,. 1 9 73 ) .. The f l uorescence e xc i tation and emi s s ion curve s o f ceroid and l ipofusc i n a r e s imi l a r though not iden t i c a l .. Lipo fus c i n h a s a n. exc i ta t io n maximum a t 360 n m a nd emi s s ion ma ximum a t 4 5 0 n m , whereas c eroid ha s e xc i ta tion maxima at 290 nm a nd 35 0 maximum at 4 35. rum .. Chio and Tappel. nm ,. and an emi s s ion. ( 19 6 9 a , 1969b) showed tha t.

(44) 29. Schi ff-bases chemi ca l l y syn the s i zed from ma lonal dehyde and amino acids or o ther a�i no compounds have f l uore scence spec tra vi r tua l l y iden t i c a l. t o tho s e o f the l ipopi gments .. They concluded that the. fluore s c ence in the synthe s i zed c ompounds as we l l as in the l ipopi gme n t s i s due t o chromopho re s with the same bas ic structure : a 1 - amino 3 - iminopropene. ( R-N=CH-CH= CH-NH-R) , formed when ma lona l dehyde c ro s s ­. l i nks w i th the pr imary amino group s o f prote ins , nucl e i c a c i d s and their b a s e s or phosphol ipid s .. S i n c e rna lona l dehyde i s a known product. of the peroxi dation of unsaturated fa tty acids , they sugge s ted that l ipop i gment fo rma t ion could be due to a di sorder of peroxida tion .. Re cent wo rk by Wo l fe et a l .. ( 1 9 7 6 , 1 9 7 7 ) l ed them to d i spute the. Schi f f -ba se n a ture o f the f luorophor e in ceroid , obta i ne d from the cerebra l cortex o f a chi l d who had d i ed from the l ate i n f a n t i l e form of NCL .. The c ero i d conta i n i n g r e s idual bodies were i so l a te d by dens i ty. grad i e n t centr i fuga t ion fol lowed by p ronase d i ge stion and showed intense fluor e scence and a typic a l u l tras tructure of curvi l inear bodi e s .. Afte r extractio n by a mixture o f chloro form and me thano l ,. 5 7 % of the d rywe i gh t o f the s torage mater ial rema ined a s a water ­ insolub l e amo rphous fluore scen t r e s idue .. According t o the author s ,. results o f i n fra -red spe c tros copy , proto n magne t ic re sonance spec tr o s copy , mas s spectrome tr y , ba s e hydrolys i s , methano l y s i s a nd ozonol y s i s , i nd i c a t ed that th e f luor e sc e n t component o f the neuron a l s to rage mate r i a l i s a retinoly comp l e x a nd i s n o t der ived from peroxid i se d polyun saturated fatty a c i d s .. In the l i gh t o f the i r. findings , the author s sugge s ted t h a t the chemical nature o f l ipofu s c i n b e r e i nves t i ga ted ..

(45) 30. Peroxidase de f i c i e n cy The f i r s t r epo r t s tha t pa tients suffe r i n g from the l a te i n fanti l e , j uven i l e and domi nant adul t forms o f NCL showed a peroxidase d e f i c i ency came from Arms trong e t al .. ( 19 7 3 ,. l 9 74 a , l 9 7 4b ) .. They found. de f i c i e n t myeloperoxid a s e activity in per iphera l granulocyte s by both h i s tochemical and spec trophotome tric methods u s i ng ben z idine and p -pheny l ened iamine as hydro gen donor s r e spective ly .. Armstrong et al .. ( 19 7 3 ) a l so found a d e c r ease o f leukocyte peroxidase activi ty i n both pare n t s o f a child w i th NCL .. Pe roxidase de f i c i ency i n patients. suffer ing from NCL has s ince be e n demon strated in seve ral ti s sues i nc l ud ing bra in , l i ve r , k idne y , thyr o id , paro t i d and submaxi l l ary sal ivary glands. (Arms trong et al . , l 9 74 c , 19 7 5 ) .. ( 19 7 5 ) a nd Awas th i e t al .. C l ausen and Jen s e n. ( 19 7 7 ) con f i rmed the reduc tion i n ac tivity. o f granulocyte peroxida s e in patients with NCL , however , the l a t t e r authors found the va l ue s o f the parents o f the i r l a te i n fa n t i l e pat ient t o be wi th i n norma l range .. On the other hand there a re a n umbe r o f publi cation s repor t i n g normal myel operoxidase activity in granulocyte s o f pati e n t s wi th NCL (An z i l et al. , 19 7 5 ; Wo l fe et al. , 19 7 6 ; Farrel l and Sum i , 1 9 7 7 ; Den Tandt and Mart in ,. 19 78 ; P i l z e t al . , 19 7 8 ) .. Normal values for. peroxidase activity were a l so reported fo r s a l i va and paro t i d s a l ivary g land in j uven i l e NC L ( Pi l z et a l . , l9 7 6 a ; P i l z and Goebe l ,. 19 7 7 ) .. When l eukocyte peroxi d a s e activity was mea s ured as so l ub l e and memb rane bound fraction s , P i l z e t a l .. ( 19 7 6b) found that in two patients w i th. the j uven i l e for m , the a c t i vi ty o f solub l e l e ukocyte pe roxi das e was con s i d e rably reduced ; i n one patient w i th the l ate infan t i l e form the a c t i vi ty was j us t b elow the norma l range and activity was normal.

(46) 31. in two pa t i e n t s with the adu l t form .. In a l l patien t s , the a c t ivity. of membrane -bound l e ukocyte p eroxidase wa s not sign i f i cantly a l tered . On ly one o f four obl i gate he terozygo t e s for the j uve n i l e type had d e f i c i e n t va l ue s for the so l uble enzyme .. P i l z et al .. ( l9 7 6 c ). found. no di fference be twee n the i so e l e c tr i c enzyme patte rn s o f l e ukocyte peroxi dase be tween patients wi th NCL and norma l contro l s .. In a s tudy o f granul ocyte en zyme activities i n pati e n t s w i th j uven i l e NCL , Tsan e t al .. ( 19 7 8 ). found p -phenyl ened i ami ne to be. un suitabl e as hydrogen donor for the s tudy o f peroxi dase .. Using. guiacol a s hydro gen donor they found e i ther norma l o r reduced peroxidase ac tivi ty i n the i r patient s . showed normal a c t ivi t i e s .. Arms trong D.. A l l other enzyme s s tudied (pe r s . comm. ). points out that. l e uko cyte p eroxi dase de f i c i ency can be shown only whe n c e t r imide ( techn i con WBC d i l ue n t , cat . no . TO l - 0 4 8 6 - 10 . Bri j -3 5 per l i tr e of cetrimi de ). Add 1 ml Te chni con. is used to l y s e ertythro cyt e s , i n. the pur i f i ca t i on proce s s o f the l eukocyte s .. The rea son for thi s. phenomenon i s not k nown , but the cetrimide d i l uent s o l ut io n conta i n s 0 . 1 % formal dehyde , and th i s addit ive po s s ibly has an e f fe c t o n the stab i l i ty o f the membrane bound p eroxidase ( Jo l l y R . D . , p e r s . comm. ) . Of the author s quo ted above , who found normal leukocyte peroxidase activi t i e s in patients w i th NCL, only Farre l l and Sumi. ( 19 7 8 ) They. reported o n the use o f 0 . 2 % cetr imide i n one o f the i r a s says .. described i t s e ffect a s 1' solubi l i z in g the pe roxidase a c t i v i ty from whole l eukocyte s " .. An. a dd i t ional p eroxid a s e d e f i c iency has been d e s c r ibed by. We stermarck and Sandholm ( 19 7 7 ). and Jen s e n e t al.. ( 19 7 8 ) .. The.

(47) 32. former found a decrea sed e rythrocyte glu tathione peroxida s e activity in th ree pat i e n t s w i th infan t i l e and ni ne pat i e n t s w i th the j uve n i l e form o f NCL . value s .. Se l e n ium suppl ementation resul ted i n a re turn to normal. Jen sen e t al .. ( 1 978). con f i rmed the reduc ed e rythrocyte. gl utath ione pe rox i d a s e a c t iv i ty in a group o f 1 0 pat ients w i th Batten ' s syndrome .. Using d i f ferent peroxide donors , the i r pa tients could be. divided i n to two groups .. One group showed reduced activity u s ing hydrogen. pe roxide or cumene hydroperoxide a s dono rs , the o ther showed an increased ac tiv i ty when t-butyl hydroperoxide wa s used .. The author s a l so reported. an inve r s e r e l a t io n s h ip b e twee n erythro cyte glutathione peroxidase activ i ty and se rum e i cosatrienoic ac id content i n the i r pa t i ents .. S e l e n i um. content o f erythroc yte s was no rmal , but va lues for who l e blood were reduced when compared with control s .. Jensen e t a l.. ( 19 7 7 ). found norma l. or eve n i ncreased l eve l s o f g lutathione pe rox idase a c t i v i ty in se rum from patients with the l a t e in fan t i l e form o f NCL .. Acid pho sphata se a c t iv i t y I n a s tudy o f ac id phosphatase activ ity i n granulocyt e s and lymphocytes of pa t i e n t s w i th NCL , Plum and Ni e l sen ( 1 9 7 7 ) activity i n pa t i e nt s '. found a higher. lymphocytes compared w i th norma l contro l s .. Supe rox ide d i smuta s e act ivity Marklund and P lum ( 1 9 7 8 ). found no di ffere nce i n supe roxide. d i smuta s e a c t iv i ty in l e ukocyte s and e rythrocytes of pa t i e n t s with the j uve n i l e form o f NCL , when c ompared to patients with neurological disease o ther than NCL and h e a l thy control s ..

Figure

OF FIGURES

OF FIGURES

p.10
Figure 3. 3 Transverse section of the cerebrum of an affected

Figure 3.

3 Transverse section of the cerebrum of an affected p.11
Figure 3. 8

Figure 3.

8 p.12
Figure 3 . 13

Figure 3 .

13 p.13
TABLE l. I (contd. )

TABLE l.

I (contd. ) p.25
TABLE 2 .  I

TABLE 2 .

I p.61
TABLE 3 . 1

TABLE 3 .

1 p.82
TABLE 3 .  II

TABLE 3 .

II p.92
TABLE 3 .  II (Contd . )

TABLE 3 .

II (Contd . ) p.93
TABLE 4 . 1

TABLE 4 .

1 p.141

References