American Journal of Medical Genetics 42:609-614 (1992)

Sheep and Other Animals With CeroidLipofuscinoses: Their Relevance to Batten Disease Robert D. Jolly, Ryan D. Martinus, and David N. Palmer Department of Veterinary Pathology and Public Health, Massey University, Palmerston North, New Zealand

Distinct pathological and histopathological changes distinguish the ceroid-lipofuscinoses from other storage diseases of humans and animals. These various disease entities likely reflect a variety of mutations of the same gene, or mutations of different genes associated with metabolism of the same or similar substrates. The disease in sheep most closely resembles the juvenile human disease. In it 50% of the lipopigment consists of subunit c of mitochondrial ATP synthase while the remaining constituents are considered normal for a lysosomal derived cytosome. The same subunit c has been shown to be also stored in affected English Setter, Border Collie, and Tibetan Terrier dogs, the Devon cow, and in the late infantile and juvenile human forms of disease but not in the infantile form. Thus it gives a chemical unity to at least some members of the group and allows a major conceptual change in regard to further directions of research.

KEY WORDS: dog, cattle, mitochondria1ATP synthase, subunit c, storage diseases INTRODUCTION The distinct pathological and histopathological abnormalities of the ceroid-lipofuscinoses distinguish them from other lysosomal storage diseases [Zeman and Dyken, 1969; Zeman, 19761. These include brain atrophy and the presence of intracellular fluorescent “lipopigment.” These storage pigment cytosomes have characteristic granular and lamellar ultrastructures. There is usually, though not invariably, loss of retinal photoreceptor cells. Similar diseases have been described in several domestic animals and it is to be ex-

Received for publication April 5,1991;revision received June 20, 1991. Address reprint requests to R. D. Jolly, Dept. of Veterinary Pathology and Public Health, Massey University, Palmerston North, New Zealand.

0 1992 Wiley-Liss, Inc.

pected that they reflect similar mutations to those in humans. Given the heterogeneous nature of this group of diseases, they are likely to represent a variety of mutations of the same gene, or mutations of different genes associated with the metabolism of the same or similar substrates. In humans the classification of syndromes is incomplete but up to 10 varieties are described [Dyken, 19881. Of these, the infantile, late infantile, and juvenile are the best understood and most frequently encountered entities and will be used below for comparative purposes. An adult form (Kufs disease) also occurs. Although there are clearly a number of different mutations reflected in the various syndromes, it is likely that some of the variants could be double heterozygotes of 2 mutations of the same gene. Given the evolutionary time scale since species separated from each other, it is likely that the mutations found in different species have arisen de novo in each species. They may thus each be unique in a molecular sense but can be expected to reflect the same range of metabolic errors. On the other hand the development of breeds within animal species is relatively recent and it is likely that some forms of disease within a species may reflect the same mutation. Subtle genetic differences associated with unique mutations in the various species, and overall species differences in expression of the same or similar biochemical defects, may result in minor modificationsto the clinical expression of the defect. In particular, animals have a shorter natural life span than humans and the time course of events in these diseases appears correspondingly truncated. Making accurate comparisons between species is therefore difficult. There are advantages in trying to do so but differences might also be important in resolving the biochemical pathology of the group and the normal underlying biochemistry of the metabolites concerned. This paper discusses the roles and relevance of animal models to research into the human ceroid-lipofuscinoses but with particular reference to the disease in sheep.

MODELS OF CEROID-LIPOFUSCINOSIS There are may reports of diseases resembling the ceroid-lipofuscinoses but many of these are merely case reports and incomplete in details needed to define them

610

Jolly et al. TABLE I. Findings of Interest in the Ceroid-Lipofuscinoses of the Different Species Mentioned in Text Where Subunit c Storage Has Been Investigated Species subclass Human Infantile Late infantile Juvenile Ovine South Hampshire Bovine Devon Canine English Setter Border Collie Tibetan Terrier

~

Autosomal recessive

Brain

Atrophy Retina

Yes Yes Yes

++++ +++

Yes Yes Yes

Yes

+++ ++ ++

Yes

Yes Yes Yes Yes

++

Cerebellum +

Lwol fast blue

Storage subunit c

-ve (?)

No Yes Yes

No No

+ ve + ve + ve + ve + ve + ve

Slight

+ ve

Yes

Yes Yes Yes" Yes" Yes"

"Unpublished data

more precisely. Those referred to in this paper are listed in Table I. Of these, only the English Setter dog and South Hampshire sheep have been studied extensively as models of the human group. Reports on the disease in the Border Collie [Taylor and Farrow, 19881 and Blue Heeler dogs [Cho et al., 1986; Wood et al., 19871 have noted their resemblance to the disease in the English Setter dog. Given this similarity and the relatively recent developmentof specificbreeds, it is quite likely that they reflect the same mutation. The English Setter disease has been likened to the juvenile human disease but affected dogs do not develop severe retinal degeneration with loss of photoreceptor cells so characteristic of the juvenile disease [Koppang, 1973/74, 19881. The adult subclinical form of ceroid-lipofuscinosisin Tibetan Terriers [Riis, 19921 is clearly different from the other canine forms. The diseases in sheep [Jolly et al., 1980, 1982,1988, 1989, 19901 and Devon cattle [Harper et al., 19881 are very similar and both include brain atrophy and retinal degeneration. As brain atrophy is a distinct component of the ceroid-lipofuscinosesas compared to other lysosoma1 storage diseases, it is important that it be measured or assessed in the development of any model. Brains ofthe affected English Setter have 70%ofnormal

weight [Koppang, 1973/741;those of affected sheep are only 50% and mainly associated with atrophy of the cerebral cortex [Mayhew et al., 19851. In affected sheep, necrosis of neurons, which is the basis of atrophy, follows a particular pattern beginning with laminar neuronal loss in the parietal lobe. With time, the laminar aspects become less obvious and neuronal loss extends to the occipital area and lastly affects the temporal lobes [Jolly et al., 1989; 19901. This is accompanied by an astrocytosis easily visualised by immunocytochemistry using an antibody to glial fibrillary acidic protein (GFAP) (Fig. 1).This necrosis of neurons and the concomitant astrocytosis can be followed from an early age (e.g., 2.5 months in sheep) sometime before a significant abnormal change in brain weight occurs. This relatively simple technique should be used in developing any model as it may show differences or similarities not demonstrated by other means and which might be important in fully understanding or comparing these diseases. The relative degrees of brain atrophy listed in Table I are best estimates from available information. Time-course clinical, electrophysiological, light and electron microscopic studies of retinal changes have helped an understanding of likely progressive changes in the analogous human diseases in which pathological

Fig. 1. a:A cerebral cortical gyrus from a 5 month oldlamb affected with ceroid-lipofuscinosis is stained by immunocytochemistry using a n antibody against G.F.A.P. It shows a darkened laminar area of astrocytosis which corresponds to a laminar loss of neurons. x 10. b High power of area of astrocytosis depicted in a. X 270.

Sheep and Other Animals With Ceroid-Lipofuscinoses study is essentially limited to the terminal disease stage. Some loss of vision in affected lambs can be noted at age 7 months when it is attributed to a central component. Thereafter there is also a significant retinal component and in late disease there is near complete loss of photoreceptors. Necrosis of photoreceptors is proceeded by a severe dystrophy of rod and cone outer segments which occurs prior to evidence of degeneration of the cell body. Changes in rod photoreceptor cells occur earlier and proceed more rapidly than in cone cells [Graydon and Jolly, 1984;Mayhew et al., 19851. This is reflected in degenerating a- and b-wave amplitudes of electroretinograms. In the sheep, loss of vision due to disease of the central nervous system is attributed to atrophy of the occipital cortex. This is also presumably the cause of vision loss in the English Setter dogs. In both animals there is widespread accumulation of pigment in the various cell types of the retina. In both models there is an early reduction in c-wave, interpreted in the dog as being associated with a defect in pigment epithelium [Armstrong et al., 1982; Nilsson et al., 19831. Experiments in the sheep used stimulation of c-likewaves with sodium azide and their extinction by destroying the pigment epithelium with sodium iodate [Mayhew et al., 19851. The c-wave is the residual combination of a positive change in potential amplitude generated across the pigment epithelium and a negative change in amplitude generated between the retina and vitreous [Steinberg et al., 19851. For normal sheep, this records as a residual positive change in amplitude. This combination of a positive and negative component means that a relatively small change in one component can cause a relatively major change in the combined c-wave amplitude. The implication from the data of Mayhew et al. [19851 is that there is a reduction in the trans-pigment epithelial component. It is the ability to do time-course and invasive experiments at various stages of the disease, as in the experiments mentioned above, that allows an understanding of the disease processes involved before the terminal stage is reached. Seizures are a prominent manifestation of the infantile, late infantile, and juvenile diseases and are frequently the presenting signs particularly in the first 2 diseases. This is not the case with the animal models although convulsionsare reported for the English Setter from 17 months [Koppang, 1973/741.The South Hampshire sheep develop intermittent spontaneous episodes of lip, eyelid, ear, face, head, and neck tremors which could also be induced by handling them. These tremors, which are abolished by intravenous phenobarbitone or diazepam, are partial symmetrical seizures that do not generalise [Mayhew et al., 19851. Sheep have a high seizure threshold so these signs probably equate with the more severe and generalised tremors in affected children. This is a good example of a species difference modifying the clinical expression of a particular disease. Seizures can eventually occur in the sheep but usually when death is imminent. Given the species differences in clinical expression and the truncated course of the disease associated with their shorter life span, the disease in sheep clinically

611

and pathologically most closely resembles the juvenile human entity. CHEMICAL STUDIES ON ISOLATED LIPOPIGMENT Inborn errors of metabolism are reflected by elevation of the deficient enzyme’ssubstrate or derivatives of it, a principle first enunciated by Garrod in 1908. In the case of storage diseases, the dominantly stored species should therefore reflect the underlying biochemical anomaly. It is analytical studies of stored material that has led to the elucidation of the biochemical defect in most storage diseases. The feasibility of isolating and solubilising lipopigment from ceroid-lipofuscinosis brains was demonstrated in the early 1970s [Siakotos et al., 19721. With the exception of studies by Wolfe et al. [1977,19811and Ng Ying Kin [19831this approach has not been widely adopted with human tissues. Research into the sheep model though has been driven by the principle discussed above. The availability of an easily bred and maintained animal in adequate numbers on a year-round basis has facilitated such analytical studies. The isolation technique has been greatly simplified and now depends on homogenisation, osmotic shock, sonification, and differential centrifugation [Palmer et al., 1986a,b,19881. In most cases relatively pure preparations of lipopigment are obtained but isolation from frozen tissue is less satisfactory and an additional CsCl isopycnic centrifugation step is often necessary. The ability to commence isolations minutes after euthanasia also has the advantage that artifactual post-mortem changes are minimised. All the major chemical constituents of this so-called lipopigment have been reported and discussed elsewhere but are summarised in Figure 2. The major stored species is the dicyclohexylcarbodiimide(DCCD)binding proteolipid, subunit c of the complex mitochondrial ATP synthase oligomeric protein. This accounts for a t least 50%of the pigment mass. All the other components are normal for a lysosomal-derived cytosome and no other mitochondrial components are stored [Palmer et al., 1985,1989,1990;Fearnley et al., 1990;Hall et al., 19891. Subsequent studies on the bovine, canine (3 breed forms), and human lipopigments (late infantile and juvenile diseases) have shown similar storage of subunit c (Table I) [Palmer et al., 1990; Martinus et al., 1991, and unpublished]. Because this is the only major species stored, the biochemical defects should relate to its metabolism. As such the animal forms are analogous models of a t least 2 of the human entities. In contrast patients with the infantile form of ceroid-lipofuscinosis do not store subunit c but rather another protein [Palmer et al., 19921. Subunit c of mitochondrial ATP synthase can be extracted with lipids by chlorofondmethanol mixtures. For this reason it is known as a proteolipid, a term introduced by Folch and Lees [19511. It does not necessarily imply that the protein has covalently attached lipid. Subunit c also stains poorly with Coomassie blue. These unusual properties may help explain why this type of protein was not recognised as being associated with the ceroid-lipofuscinoses in the past. The early

612

Jolly et al.

continued implication of lipid peroxidation [Jolly and Dalefield, 19901. The same argument can be made for the descriptive name “ceroid-lipofuscinosis”by which the group is commonly designated. Although we have used the descriptive term “proteolipid proteinosis” [Palmer et al., 1985; Jolly et al., 19901which is more accurate, we would hesitate to suggest a widespread name change a t this stage when a more definitive biochemical classification is perhaps imminent and when the number of eponyms and name changes over the last century have already sufficiently confused the issue. Nevertheless a subgroup of “subunit c storage diseases” is now clearly established.

INTERPRETATION OF HISTOCHEMISTRY AND ULTRASTRUCTURE

Dolichol- P - P - Oligosaccharides Fig. 2. Schematic representation of proportions of the main chemical species in pigment isolated from sheep with ceroid-lipofuscinosis.

isolation and analytical studies of Siakotos et al. [1972] noted that isolated “ceroid” was soluble in lipid solvents and that almost half of this was “acidiclipid polymer.” It was proposed then that this polymer was the “stored substance” in the neuronal ceroid-lipofuscinoses. In retrospect, it seems likely that the designated “acidic lipid polymer” was the proteolipid subunit c of mitochondria1 ATP synthase. In view of the above analyses, the lipid peroxidation hypothesis [Zeman, 1974; Armstrong et al., 19741is no longer tenable for the disease in sheep. The same is probably true for other forms of ceroid-lipofuscinoses in humans and animals where storage of subunit c is a feature (Table I). Quantitative studies on dolichol and dolichol P-P linked oligosaccharides [Palmer et al., 1986a; Hall et al., 19891 show that they are relatively minor stored components and compatible with lysosoma1 derivation of the pigment cytosomes. Thus, they are secondary phenomena. For many years the lipopigment in the ceroid-lipofuscinoses was referred to as “ceroid”because of similar staining and fluorescent properties t o “ceroid” originally described in cirrhotic livers and vitamin E deficiency [Porta and Hartcroft, 19691. With this background there was an associated postulate that pigmentogenesis was primarily associated with oxidation of lipids. The pathogenesis of pigment in ovine ceroid-lipofuscinosis and, by comparison and implication, other forms of the disease, is now distanced from the pathogenesis of “ceroid” as originally conceived. Thus, it is no longer an appropriate description to use in connectionwith Batten and analogous diseases. To do so for this or other pathological pigments of uncertain origin is unnecessary as well as misleading due to the

The characteristic storage cytosomes in the ceroidlipofuscinoses are fluorescent and stain for lipids. They are usually also PAS positive and stain strongly with luxol fast blue (Fig. 3). These are the histochemical and physical characteristics that most clearly define this group of pigments. There has been no uniformly acceptable explanation for fluorescence of these pigments. Palmer et al. H986aI have suggested that fluorescence may be a property of the interaction of the stored protein and its peculiar lipid environment, a suggestion supported by reconstitution of fluorescent cytosomes from non-fluorescent purified subunit c and phospholipids (Palmer et al., unpublished). The various lipid stains can be expected to react with neutral lipids and phospholipids present but may also react with the hydrophobic proteolipid subunit c. This is particularly likely with paraffin-embedded sections in which pigment still stains strongly with Sudan black. In fact this stain is preferred for early diagnosis by brain biopsy. It can unequivocally allow diagnosis in affected lambs that die a t birth. Luxol fast blue is considered a stain for phospholipids and phospholipid/protein complexes in fixed tissues [Pearse, 19851. In neuropathology it is used as a stain for

Fig. 3. Neuron from a sheep with ceroid-lipofuscinosis: parafin section, luxol fast blue. x 900.

Sheep and Other Animals With Ceroid-Lipofuscinoses myelin even in paraffin block sections. An intrinsic protein of myelin is the prototype proteolipid and it is likely that it is this type of molecule that stains in myelin and storage bodies in paraffin block sections. In Table I, strong luxol fast blue staining is a characteristic of all but one disease listed. Although luxol fast blue staining of cytosomes may not necessarily be specific for this disease, it is probably an important characteristic of those storing subunit c or perhaps similar proteolipids. Of note are the observations that in the infantile disease, cytosomes do not stain with luxol fast blue [Lake, 19841 as in the late infantile or juvenile diseases and subunit c is not stored [Palmer et al., 19901.A different protein is stored in this very early onset disease (see above and Palmer et al. [19901).However, where there is myelin degeneration as a result of neuronal loss, phagosomes in macrophages can be expected to stain with luxol fast blue because of endocytosed myelin debris.

613

mitochondrial genes [Walker et al., 19911. Little is known of the control factors that allow organised synthesis and assembly of both nuclear and mitochondrial gene products into this important oligomeric protein complex. This is now an added raison d’6tre for continued research into ceroid-lipofuscinosis. Over a period of more than 20 years when a great many lysosomal storage diseases have been defined in biochemical terms, the ceroid-lipofuscinosesstood alone as an enigma. Their elucidation in biochemical terms now appears imminent through studies on the sheep model and the recognition that their pathogenesis probably reflects anomalies of metabolism affecting subunit c of mitochondrial ATP synthase. ACKNOWLEDGMENTS This research was funded by the United States Department of Health and Human Services NIH Grant No. NS11238. We would also acknowledge the help of nuDISCUSSION merous colleagues who have contributed to research The recognition that subunit c, a proteolipid, is the referred to in this paper and whose names are listed dominantly stored species rather than peroxidised lipid/ with our own in the references listed. protein polymers as originally perceived allows a major REFERENCES conceptual change in the approach to ceroid-lipofuscinosis research. Many of the characteristics of the Armstrong D, Dimmitt S, Van Wormer DE (1974): Studies in Batten disease. I. Peroxidase deficiency in granulocytes. Arch Neurol pigment and other enigmatic observations are ex30144-152. plained by this observation. The common storage of this Armstrong D, Koppang N, Nilsson SE (1982): Canine hereditary proteolipid in some other animal and human forms of ceroid-lipofuscinosis. Eur Neurol 21:147-156. the disease (Table I) gives a chemical unity to the group Cho D-Y, Leipold HW, Rudolph R (1986): Neuronal ceroidosis (Ceroidlipofuscinosis) in a blue heeler dog. Acta Neuropathol (Berl) previously missing. However, the infantile disease is 69~161-164. different implying that not all diseases falling within the general classification of ceroid-lipofuscinosisare as Dyken P (1988): Reconsideration of the classification of the neuronal ceroid-lipofuscinoses. Am J Med Genet [Suppll 569-84. closely related. It is important to continue analysis of Fearnley IM, Walker J E , Martinus RD, Jolly RD, Kirkland KB. Palmer isolated pigment so that subclassifications according to DN (1990): The sequence ofthe major protein stored in ovine ceroiddominantly stored species can be made. lipofuscinosis is identical to that of the DCCD-reactive proteolipid of mitochondrial ATP synthase. Biochem J, 268:751-758. There are at least 2 genetically distinct human forms storing subunit c, i.e., the late infantile and juvenile Folch J, Lees M (1951): Proteolipides, a new type oftissue lipoproteins. J Biol Chem 191:807-817. disease. There are many instances in other lysosomal AE (1908): In born errors of metabolism (Croonian lectures) storage disease where a series of mutations affecting one Garrod Lancet 2(i):73, 142, 214. Cited by The Editors (1972): Inherited or more genes result in storage of the same substrate, variation and metabolic abnormality. In StanburyJB, Wyngaarden JB, Fredrickson DS (eds): “The Metabolic Basis of Inherited Dise.g., GM, ganglioside [Neufeld, 19891. If 2 or more mutaease.” New York: McGraw-Hill Book co., pp 3-27. tions affect the same gene, then it is to be expected that RJ,Jolly RD (1984): Ceroid-lipofuscinosis (Batten’s disease): Graydon intermediate diseases would be associated with double Sequential electrophysiologic and pathologic changes in the retina heterozygosity. Such has not clearly been demonstrated of the ovine model. Invest Ophthalmol Vis Sci 25:294-301. in any of the ceroid-lipofuscinoses although the large Hall NA, Jolly RD, Palmer DN, Lake BR, Patrick AD (1989):Analysis number of subtypes suggest it. Careful collection of data of dolichyl pyrophosphoryl oligosaccharides in purified storage cytosomes from ovine ceroid-lipofuscinosis. Biochim Biophys Acta and genealogy analyses may help resolve this aspect; 993:245-251. animal breeding studies may also be informative. The Harper PAW, Walker KH, Healy PJ, Hartley WJ, Gibson AJ, Smith JS likelihood of forms of ceroid-lipofuscinosiswithin a spe(1988): Neurovisceral ceroid-lipofuscinosis in blind Devon cattle. cies reflecting a single mutation has been mentioned Acta Neuropathol (Berl) 75632-636. above. Any “within species” breeding studies should Jolly RD, Delefield RR (1990): Lipopigments in veterinary pathology: Pathogenesis and terminology. In Porta EA (ed): “Lipofuscin and thus use clearly distinct forms of the disease within that Ceroid Pigments.” New York Plenum Publishing Corp., in press. species. A t this stage this is likely to be possible only Jolly RD, Janmaat A, West DM, Morrison I (1980): Ovine Ceroidwith the dog but before this is meaningful, careful comlipofuscinosisamodel of Batten’s disease. Neuropathol Appl Neuparative studies need to be made. robiol6:195-209. Research into the ceroid-lipofuscinoses is mainly Jolly RD, Janmaat A, Graydon R J , Clemett RS (1982): Ceroid-lidriven by the need to understand their pathogenesis so pofuscinosis (Batten’s disease). In Armstrong D, Koppang N, Rider JA (eds): “Ceroid-lipofuscinosis: The Ovine Model.” Amsterdam: that better diagnostic, control, or therapeutic strategies Elsevier Biomedical Press, pp 219-228. can be developed. It should also be recognised that their Jolly RD, Shimada A, Craig AS, Kirkland KB, Palmer DN (1988): elucidation is likely t o help understand the general biolOvine ceroid-lipofuscinosis I1 Pathologic changes interpreted in ogy of the mitochondrial ATP synthase complex. This light of biochemical observations. Am J Med Genet [Suppll5159170. contains 14 proteins, which are coded by nuclear and

614

Jolly et al.

Jolly RD, Shimada A, Dopfmer I, Slack PM, Birtles MJ, Palmer DN (1989): Ceroid-lipofuscinosis (Batten’s disease): Pathogenesis and sequential neuropathological changes in the ovine model. Neuropathol Appl Neurobiol 15371-383. Jolly RD, Martinus RD, Shimada A, Fearnly IM, Palmer DN (1990): Ovine ceroid-lipofuscinosis is a proteolipid proteinosis. Can J Vet Res 54:15-21. Koppang N (1973174):Canine ceroid-lipofuscinosis. A model for human neuronal ceroid-lipofuscinosis and ageing. Mech Ageing Dev 2:421-445. Koppang N (1988): The English Setter with ceroid-lipofuscinosis: A suitable model for the juvenile type of ceroid-lipofuscinosis in humans. Am J Med Genet [Suppll 5117-125. Lake BD (1984): Lysosomal Deficiencies. In Adams JH, Corsellis JA, Duchen LW (eds): “Greenfields Neuropathology.” London: Edward Arnold, pp 491-572. MartinusRD,HarperPAW,Jolly RD, Bayliss SL, Midwinter GG, Shaw GJ, Palmer DN (1991): Bovine ceroid-lipofuscinosis (Batten disease): The major component stored is the DCCD reactive proteolipid, subunit c of mitochondrial ATP synthase. Vet Res Commun 1585-94. Mayhew IG, Jolly RD, Pickett BT, Slack PM (1985): Ceroid-lipofuscinosis (Batten’s disease): Pathogenesis of blindness in the ovine model. Neuropathol Appl Neurobiol 11:273-290. Neufeld EF (1989):Natural history and inherited disorders of a lysosoma1 enzyme P-Hexosaminidase.J Biol Chem 264:10927-10930. Ng Ying Kin NMK, Haltia JP, Wolfe LS (1983):High levels of brain dolichols in neuronal ceroid-lipofuscinosis and senescence. J Neurochem 40:1465-1473. Nilsson SS, Armstrong D, Koppang N, Persson P, Milde K (1983): Studies on the retina and the pigment epithelium in hereditary canine ceroid-lipofuscinosis. Invest Ophthalmol Vis Sci 24:77-84. Palmer DN, Husbands DR, Jolly RD (1985):Phospholipid fatty acids in brains of normal sheep and sheep with ceroid-lipofuscinosis. Biochim Biophys Acta 834:159-163. Palmer DN, Husbands DR, Winter PJ, Blunt JW, Jolly RD (1986a): Ceroid-lipofuscinosis in sheep. I. Bis(monoacylglycero)phosphate, dolichol, ubiquinone, phospholipids, fatty acids and fluorescence in liver lipopigment lipids. J Biol Chem 261:1766-1772. Palmer DN, Barns G, Husbands DR, Jolly RD (1986b): Ceroid-lipofuscinosis in sheep: 11. The major component of lipopigment in liver, kidney, pancreas and brain is low molecular weight protein. J Biol Chem 261:1773-1777. Palmer DN, Martinus RD, Barns G, Reeves RD, Jolly RD (1988):Ovine ceroid-lipofuscinosis I Lipopigment composition is indicative of a lysosomal proteinosis. Am J Med Genet [Suppll 5141-158. Palmer DN, Martinus RD, Cooper SM, Midwinter GG, Reid JC, Jolly RD (1989):Ovine ceroid-lipofuscinosis: The major lipopigment pro-

tein and the lipid binding subunit of mitochondrial ATP synthase have the same NH, terminal sequence. J Biol Chem 26457365740. Palmer DN, Jolly RD, Martinus RD (1992):Ovine ceroid-lipofuscinosis: Analyses of isolated lipopigment. Am J Med Genet (this issue). Pearse AGE (1985):“Histochemistry, Theoretical and Applied. Vol. 2. Analytical Technology.”New York Churchill Livingston, 4th edition, pp 686-690. Porta EA, Hartcroft WS (1969): Lipid pigments in relation to ageing and dietary factors (Lipofuscins). In Wolman M (ed): “Pigments in Pathology.” London: Academic Press Inc., pp 191-235. Riis RC (1992):The central nervous system changes in a young adult Tibetan Terrier with subclinical neuronal ceroid-lipofuscinosis. Am J Med Genet (this issue). Siakotos AN, Goebel HH, Pate1 V, Watanabe I, Zeman W (1972):The morphogenesis and biochemical characteristics of ceroid isolated from cases of ceroid-lipofuscinosis. In Volk BW, Aronson GM (eds): New York: Plenum Publishing Corporation, pp 53-61. Steinberg RH, Linsenmeier RA, Grief ER (1985): Retinal pigment epithelial cell contributions to the electroretinogram and electrooculogram. In Osborne NM, Chader GJ (eds): “Progress in Retinal Research.” New York Pergamon vol4, pp 44-66. Taylor RM, Farrow BRA (1988):Ceroid-lipofuscinosis in Border Collie dogs. Acta Neuropathol (Berl) 75627-631. Walker J E , Lutter R, Dupuis A, Runswick MJ (1991):Identification of the subunits FIFOATPase from bovine heart mitochondria. Biochemistry, 305369-5378. Wolfe LS, Ng Ying Kin NMK, Baker RR, Carpenter S, Andermann F (1977): Identification of retinoyl complexes as the autofluorescent component of the neuronal storage material in Battens disease. Science 195:1360-1362. Wolfe LS, Ng Ying Kin NMK, Baker RR (1981): Batten disease and related disorders. New findings on the chemistry of storage material. In Callahan JW, Lowden JA (eds): “Lysosomes and Storage Diseases.” New York Raven Press pp 315-330. Wood PA, Sisk DB, Styer E, Baker H J (1987):Animal Model: Ceroidosis (Ceroid-lipofuscinosis) in Australian cattle dogs. Am J Med Genet 26:891-898. Zeman W (1974):Studies in the neuronal ceroid-lipofuscinoses. J Neuropathol Exp Neurol 33:l-12. Zeman W (1976): The neuronal ceroid-lipofuscinoses. In Zimmerrnan HM (ed): “Progress in Neuropathology,”Vol. 111. New York: Grune and Stratton, pp 207-223. Zeman W, Dyken P (1969): Neuronal ceroid-lipofuscinosis (Batten’s disease): Relationship to amaurotic family idiocy? Pediatrics 44:570-583.