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

(1)Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author..

(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 ..