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Neuronal ceroid-lipofuscinosis in a domestic cat: Clinical, morphological and immunohistochemical findings
-j
J, Comp. Path. 1997 Vol. ll7, 17 24
C F Neuronal Ceroid-lipofuscinosis in a Domestic Cat: Clinical, Morphological and Immunohistochemical Findings H. W e i s s e n b 6 c k a n d C. R 6 s s e l * Institute of Pathology and Forensic Veterina~ Medicine, Universi~ of Veterinary Medicine, Josef Baumann-Gasse 1, A-1210 Nenna, and *Small Animal Clinic Breitensee, Breitenseer Strasse 16, A-1140 Vienna, Austria
Summary A 9-month-old domestic shorthair cat was humanely killed because of uncoordinated gait, myoclonus, seizures and reduced vision. Histological, immunohistochemical and ultrastructural examination revealed a neuronal storage disease consistent with neuronal ceroid-lipofuscinosis (NCL). Neurons contained Sudan black- and luxol fast blue-positive material which was autofluorescent. Immunohistochemically, the storage material was found to contain subunit c of mitochondrial ATP synthase, a protein recently recognized as the main component of the storage material in NCL. Ultrastructurally, the material consisted of curvilinear and fingerprint bodies, which are indicative of NCL. 9 1997W.B. Saunders Company Limited
Introduction Neuronal ceroid-lipofuscinoses (NCLs, Batten's disease) represent a group of recessively inherited neurodegenerative disorders (Kohlschiitter et al., 1993; Jolly, 1995). Morphologically, they are characterized by brain atrophy, diffuse loss of neurons with resulting gliosis, and lysosomal storage of autofluorescent, Sudan black-, luxol fast blue (LFB)- and sometimes periodic acid-Schiff(PAS)positive material in neurons and glial cells (Jolly et al., 1989; Koppang, 1992). Ultrastructurally, the storage material is composed of so-called curvilinear and fingerprint bodies (Jolly et al., 1989; Palmer et al., 1992). Usually, the storage is not restricted to the central nervous system and can be found in a ~/ariety of extraneural tissues. Unlike other lysosomal storage diseases (e.g., gangliosidosis, mannosidosis, glycogenosis and sphingomyelinosis), which are clearly defined by deficiencies of degrading enzymes and lysosomal accumulation of the specific substrate, N C L is still poorly understood in respect of the underlying biochemical defect, and the exact composition of the storage material has only recently been clarified. Jolly et al. (1990) and Palmer et al. (1992) demonstrated in the New Hampshire sheep model that the storage material consisted primarily of a hydrophobic protein, the subunit c of mitochondrial A T P synthase. NCLs are divided into five different forms in man (Kohlschtitter et al., 1993). In one form the genetic defect is associated with chromosome 1, and in a second form with chromosome 16 (Kohlschiitter et al., 1993). Two important 0021-9975/97/050017+08 $12.00/0
9 1997 W.B. Saunders Company Limited
18
H. Weissenb6ck and C. R6ssel
models, maintained in breeding colonies, are the English setter (Koppang, 1992) and the New Hampshire sheep (Jolly et al., 1989). NCLs have been described in a variety of breeds of dog (Westlake et al., 1995), in Devon cattle (Harper et al., 1988), Rambouillet sheep (Edwards et al., 1994), Nubian goats (Fiske and Storts, 1988), a cynomolgus monkey (Jasty et al., 1984) and mutant mice (Pardo et al., 1994). There are three reports of N C L in cats (Green and Little, 1974; Nakayama et al., 1993; Bildfell et al., 1995), two from Canada (two Siamese cats and a domestic shorthair cat) and one from J a p a n (a domestic cat). This is the first report of N C L in a cat in Europe. The diagnosis was supported by immunohistochemical demonstration of subunit c of mitochondrial ATP synthase. Materials and Methods
Histopathology and Fluorescence Microscopy At necropsy, samples of brain, spinal cord, liver, spleen, kidney, heart and lung were collected and fixed in 7% buffered formalin. The tissues were dehydrated through graded alcohols and, after a xylene step, embedded in paraffin wax and sectioned at 5 gm. The following stains were applied to sections of the central nervous system (CNS): haematoxylin and eosin (HE), periodic acid-Schiff (PAS), Sudan black, luxol fast blue (LFB), Nile blue A and cresyl echt violet. Frozen sections of formalin-fixed material were stained with Sudan III and Sudan black. Unstained frozen sections of formalin-fixed material and unstained dewaxed paraffin-wax sections, as well as HEstained sections, were examined with the fluorescence microscope (excitation filter 450 490 nm, barrier filter 520 rim). Extraneural tissues were stained with HE and examined by light and fluorescence microscopy.
Immunohistochemistry Rabbit antibodies to glial fibrillary acid protein (GFAP) (Sigma, St Louis, USA; dilution 1 in 800) and to subunit c ofmitochondrial ATP synthase (produced according to Palmer et al., 1995; dilution 1 in 1000) were used. Dewaxed sections were incubated with 10% normal goat serum (NGS) for 30min to reduce background staining. Endogenous peroxidase activity was blocked by incubation with 2% H202. The sections were incubated for 16 h at 4~ the primary antibodies being diluted in phosphate-buffered saline (PBS) solution containing NGS 1%. Subsequent steps were made with the Vectastain| ABC-kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. Brain material from a sheep with NCL served as a positive control for staining of subunit c, and normal cat brain as a negative control.
Electron Microscopy (EM) Formalin-fixed cubes (1 mm 3) from the medulla oblongata were washed for 24 h in PBS, postfixed with osmium tetroxide and embedded in Epon according to standard techniques. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 400 T transmission electron microscope. Results
Clinical History A 9-month-old neutered male cat (European domestic shorthair) had a 2week history of uncoordinated gait and episodes of myoclonus and seizures.
N e u r o n a l G e r o i d - l i p o f u s c i n o s i s in a Gat
19
Clinical examination revealed generalized ataxia and strongly reduced postural reactions ("wheelbarrowing" and extensor postural thrust reactions). Spinal reflexes and radiographs were normal. Serological examinations for toxoplasmosis, feline infectious peritonitis, and feline immunodeficiency virus infection produced negative results. The cat was feline leukaemia virusnegative. Treatment for 2 weeks with prednisolone, vitamin-B complex and phenobarbital failed to bring about a clinical improvement, and the animal was unable to recognize food and had severely reduced vision with normal pupillary reflex. At the owner's request the cat was humanely destroyed.
Macroscopical Findings, Histopathology and FluorescenceMicroscopy Marked atrophy of the brain, particularly of the cerebral hemispheres, and a moderate enlargement of the lateral ventricles were noted (Fig. 1). Histologically, an increased cellularity of the cerebral grey matter due to an increased number of glial cells in association with reduced numbers of neurons was seen. The subpial region of the cerebrum showed spongy degeneration and complete loss of neurons. In the cerebellum there was widespread loss of Purkinje cells, which were largely replaced by glial cells located between the molecular and granular layers (Fig. 2). Neurons in all regions of the brain contained single or multiple cytoplasmic inclusions, which were transparent with cresyl echt violet stain, and either transparent or eosinophilic with HE stain (Fig. 3A). Large quantities of storage material were located in the brain-stem nuclei and grey matter of the spinal cord. Some neurons containing large quantities of storage material had undergone lytic necrosis. Storage material stained black with Sudan black in both paraffin-wax and frozen sections. With Nile blue A and LFB, it stained deep .blue (Fig. 3B) and part of the material was weakly pink with PAS. Illumination by ultraviolet light showed bright yellow autofluorescence of unstained frozen and paraffin-wax sections and of HE-stained paraffin-wax sections (Fig. 3C). None of the extraneural tissues examined showed any detectable accumulation of autofluorescent storage material.
Immunohistochemistry GFAP-immunohistochemistry showed marked proliferation of reactive astrocytes in the cerebral hemispheres (Fig. 4) and the granular and Purkinje cell layers of the cerebellum. Subunit c was demonstrated in the cytoplasm of many neurons (Fig. 5) and glial cells. Specific staining was particularly intense in thalamic nuclei, vestibular nuclei and Purkinje cells. In other regions of the brain and in the spinal cord the staining reaction was comparatively weak.
Electron Microscopy (EM) Ultrastructural examination revealed many secondary lysosomes in the cytoplasm of neurons, containing multilamellar arrays consistent with curvilinear
20
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H. Weissenb6ck and C. R6ssel
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o
Fig. 1. Transverse section of the atrophic brain of the cat with neuronal ceroid-lipofuscinosis (right), compared with that of a brain from an age-matched cat without neurological disease (left). Fig. 2. Cerebellum with widespread loss of Purkinje cells. The only remaining Purkinje cell is marked by an arrow. HE. x 200. Fig. 3. (A) Neurons with cytoplasmic storage of granular, slightly eosinophilic material (arrows) in the mesencephalon. HE. x 500. (B) Neuronal storage material is stained deep blue with LFB. x 500. (C) Bright yellow autofluorescence of the cytoplasmic storage material. HE, fluorescence micrograph (excitation filter 450 490 nm, barrier filter 520 nm). x 350.
bodies or fingerprint structures. The curvilinear bodies were composed of several (average three) parallel lamellar profiles with a periodicity of 6 nm (Fig. 6A). In some of the bodies the lamellae numbered as many as 10, with an underlay of medium electron-dense homogeneous material. The fingerprint structures showed medium electron density in low magnification. A homogeneous, electron-dense matrix was interspersed with multiple parallel lamellar profiles (periodicity 6 nm) arranged in a fingerprint-pattern (Fig. 6B).
Neuronal
Fig. 4.
Fig. 5.
Geroid-lipofuseinosis
in a Cat
21
(A) Increased number of astrocytes in the cerebral cortex of the cat with neuronal ceroidlipofuscinosis, and (B) normal number of astrocytes in the cerebral cortex of a cat without neurological disease. GFAP-immunohistochemistry. x 85. Subunit c-immunoreactivity of the" cytoplasmic storage material in Purkinje cells and (inset) a neuron from a thalamic nucleus. Immunohistochemistry for subunit c of mitochondrial ATP synthase, x 360. Inset: differential interference contrast, x 500.
22
Fig. 6.
H . Weissenb6ck and G. R 6 s s e l
Curvilinear (A) and fingerprint (B) patterns of the storage material. EM. x 66 000 (A). x 108 000
(B). Discussion
The present case resembled in many ways cases of NCL already reported in man and animals. Thus, the clinical signs (myoclonus, seizures, reduced postural reactions and loss of vision) resemble those reported in man and in the English setter. In sheep, blindness is the major clinical sign, and seizures arid myoclonus have not been reported. In the three previous reports of feline cases, the animals were aged 7-22 months at the onset of disease. Our cat was of comparable age (9 months). The clinical presentations, however, differed. Nakayama et al. (1993) described only shivering and difficulty in walking. Green and Little (1974) and Bildfell et al. (1995) reported convulsions and hyperaesthesia, but no gait abnormalities. Blindness was found only by Bildfell et al. (1995). The major clinical signs in our case were generalized ataxia, reduced postural reactions, seizures, myoclonus and reduced vision. Brain atrophy, as demonstrated by Jolly et al. (1990) in sheep, was also present in our case and in the cat described by Nakayama et al. (1993). The main histological features in the present case (loss of neurons, gliosis and granular neuronal cytoplasmic inclusions) accorded with those of previous reports of NCL in various species. The histochemical staining properties and autofluorescence of the inclusions were indicative of NCL. In most forms of human NCL, the inclusions have a characteristic ultrastructure (curvilinear and fingerprint bodies), also noted in our case. In the cat described here, as in the feline cases of Green and Little (1974) and Bildfell et al. (1995), autofluorescent storage material was not found outside the CNS. Nakayama et al. (1993), however, found storage material in the
Neuronal Ceroid-lipofuscinosis in a Cat
23
mononuclear phagocyte system of the liver, spleen and lymph nodes, in addition to the CNS. Recently, more than half of the storage material in ovine NCL was found to be composed of a hydrophobic protein, the subunit c of mitochondrial ATP synthase (Jolly et al., 1990; Palmer et al., 1992). This protein was also found in several human forms of NCL (Westlake et al., 1995), some canine forms (Westlake et al., 1995), a bovine form (Martinus et al., 1991) and in a "mouse model" (Pardo et al., 1994). It is generally accepted that lysosomal accumulation of subunit c is a cardinal feature in the pathogenesis of NCL. The exact biochemical mechanism leading to the storage is still unknown. A mitochondrial rather than a lysosomal defect may result in accumulation of complexed material that cannot be adequately catabolized (Jolly, 1995). Katz and Siakotos (1995) demonstrated that a specific methylation of one of the lysine residues of subunit c plays an important role in its lysosomal storage. In none of the previously reported feline cases has demonstration ofsubunit c been attempted. Immunohistochemical labelling gave a positive result in the cat described here, which therefore shared an important feature seen in human late infantile NCL and juvenile NCL and in some well defined NCLs in animals. Acknowledgments
The authors thank S. L. Bayliss, Massey University, New Zealand and D. N. Palmer, Lincoln University, New Zealand, for kindly providing antiserum to subunit c and positive control tissue, and for valuable advice. We are grateful to I. Friedl, K. Kain, E. Fitscha and K. Fragner for skilful technical assistance and to K. Bittermann for excellent photographic work. References
Bildfell, R., Matwichuk, C., Mitchell, S. and Ward, P. (1995). Neuronal ceroidlipofuscinosis in a cat. VeterinaryPathology, 32, 485-488. Edwards, J. F., Storts, R. W.,Joyce, J. R., Shelton, J. M. and Menzies, C. S. (1994). Juvenile-onset neuronal ceroid-lipofuscinosis in Rambouillet sheep. Veterinary Pathology, 31, 48 54. Fiske, R. A. and Storts, R. W. (t 988). Neuronal ceroid-lipofuscinosis in Nubian goats. Veterinary Pathology, 25, 171-173. Green, P. D. and Little, P. B. (1974). Neuronal ceroid-lipofuscin storage in Siamese cats. CanadianJournal of Comparative Medicine, 38, 207-212. Harper, P. A. W., Walker, K. H., Healy, P.J., Hartley, W.J., Gibson, A.J. and Smith, J. S. (1988). Neuroviscerat ceroid-lipofuscinosis in blind Devon cattle. Acta Neuropathologica, 75, 632-636. Jasty, V., Kowalski, R. L., Fonseca, E. H., Porter, M. C., Clemens, G. R., Bare, J. j. and Hartnagel, R. E. (1984). An unusual case of generalized ceroidlipofuscinosis in a cynomolgus monkey. VeterinaryPathology, 21, 46-50. Jolly, R. D. (1995). Comparative biology of the neuronal ceroid-lipofuscinoses (NCL): an overview. American Journal of A/ledical Genetics, 57, 307-311. Jolly, R. D., Martinus, R. D., Shimada, A., Fearnley, I. M. and Palmer, D. N. (1990). Ovine ceroid-lipofuscinosis is a proteolipid proteinosis. CanadianJournal of Veterinary Research, 54, 15 20. Jolly, R. D., Shimada, A., Dopfmer, I., Slack, P. M., Birtles, M. J. and Palmer, D. N. (1989). Ceroid-lipofuscinosis (Batten's disease): pathogenesis and sequential neuropathological changes in the ovine model. Neuropathology and Applied Neurobiology, 15, 371-383.
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H. Weissenb6ck and C. R 6 s s e l
Katz, M. L. and Siakotos, A. N. (1995). Canine hereditary ceroid-lipofuscinosis: evidence for a defect in the carnitine biosynthetic pathway. American Journal of Medical Genetics, 57, 266 271. Kohlschatter, A., Gardiner, R. M. and Goebel, H. H. (1993). Human forms of neuronal ceroid-lipofuscinosis (Batten disease): consensus on diagnostic criteria, Hamburg 1992. Journal of Inherited Metabolic Disease, 16, 241-244. Koppang, N. (1992). English setter model and juvenile ceroid-lipofuscinosis in man. AmericanJournal of Medical Genetics, 42, 599-604. Martinus, R. D., Harper, P. A. W., Jolly, R. D., Bayliss, S. L., Midwinter, G.G., Shaw, G. J. and Palmer, D. N. (1991). Bovine ceroid-lipofuscinosis (Batten's disease): the major component stored is the DCCD-reactive proteolipid, subunit c, of mitochondrial ATP synthase. VeterinaryResearch Communications, 15, 85-94. Nakayama, H., Uchida, K., Shouda, T., Uetsuka, K., Sasaki, N. and Goto, N. (1993). Systemic ceroid-lipofuscinosis in a Japanese domestic cat. Journal of Veterinary Medical Science, 55, 829-831. Palmer, D. N., Bayliss, S. L. and Westlake, V.J. (1995). Batten disease and the ATP synthase subunit c turnover pathway: raising antibodies to subunit c. American Journal of Medical Genetics, 57, 260 265. Palmer, D. N., Fearnley, I. M., Walker, J. E.; Hall, N. A., Lake, B. D., Wolfe, L. S., Haltia, M., Martinus, R. D. and Jolly, R. D, (1992). Mitochondrial ATP synthase subunit c storage in the ceroid-lipofuscinoses (Batten disease). AmericanJournal of Medical Genetics, 42, 561-567. Pardo, C. A., Rabin, B. A., Palmer, D. N. and Price, D. L. (1994). Accumulation of the adenosine triphosphate synthase subunit c in the rand mutant mouse. American Journal of Pathology, 144, 829-835. Westlake, V. J., jolly, R. D., Bayliss, S. L. and Palmer, D. N. (1995). Immunocytoehemical studies in the ceroid-lipofuscinoses (Batten disease) using antibodies to subunit c of mitochondrial ATP synthase. AmericanJournal of Medical Genetics, 57, 177 181.
I Received,November29th, 1996] Accepted,March 13th, 1997 J
J, Comp. Path. 1997 Vol. ll7, 17 24
C F Neuronal Ceroid-lipofuscinosis in a Domestic Cat: Clinical, Morphological and Immunohistochemical Findings H. W e i s s e n b 6 c k a n d C. R 6 s s e l * Institute of Pathology and Forensic Veterina~ Medicine, Universi~ of Veterinary Medicine, Josef Baumann-Gasse 1, A-1210 Nenna, and *Small Animal Clinic Breitensee, Breitenseer Strasse 16, A-1140 Vienna, Austria
Summary A 9-month-old domestic shorthair cat was humanely killed because of uncoordinated gait, myoclonus, seizures and reduced vision. Histological, immunohistochemical and ultrastructural examination revealed a neuronal storage disease consistent with neuronal ceroid-lipofuscinosis (NCL). Neurons contained Sudan black- and luxol fast blue-positive material which was autofluorescent. Immunohistochemically, the storage material was found to contain subunit c of mitochondrial ATP synthase, a protein recently recognized as the main component of the storage material in NCL. Ultrastructurally, the material consisted of curvilinear and fingerprint bodies, which are indicative of NCL. 9 1997W.B. Saunders Company Limited
Introduction Neuronal ceroid-lipofuscinoses (NCLs, Batten's disease) represent a group of recessively inherited neurodegenerative disorders (Kohlschiitter et al., 1993; Jolly, 1995). Morphologically, they are characterized by brain atrophy, diffuse loss of neurons with resulting gliosis, and lysosomal storage of autofluorescent, Sudan black-, luxol fast blue (LFB)- and sometimes periodic acid-Schiff(PAS)positive material in neurons and glial cells (Jolly et al., 1989; Koppang, 1992). Ultrastructurally, the storage material is composed of so-called curvilinear and fingerprint bodies (Jolly et al., 1989; Palmer et al., 1992). Usually, the storage is not restricted to the central nervous system and can be found in a ~/ariety of extraneural tissues. Unlike other lysosomal storage diseases (e.g., gangliosidosis, mannosidosis, glycogenosis and sphingomyelinosis), which are clearly defined by deficiencies of degrading enzymes and lysosomal accumulation of the specific substrate, N C L is still poorly understood in respect of the underlying biochemical defect, and the exact composition of the storage material has only recently been clarified. Jolly et al. (1990) and Palmer et al. (1992) demonstrated in the New Hampshire sheep model that the storage material consisted primarily of a hydrophobic protein, the subunit c of mitochondrial A T P synthase. NCLs are divided into five different forms in man (Kohlschtitter et al., 1993). In one form the genetic defect is associated with chromosome 1, and in a second form with chromosome 16 (Kohlschiitter et al., 1993). Two important 0021-9975/97/050017+08 $12.00/0
9 1997 W.B. Saunders Company Limited
18
H. Weissenb6ck and C. R6ssel
models, maintained in breeding colonies, are the English setter (Koppang, 1992) and the New Hampshire sheep (Jolly et al., 1989). NCLs have been described in a variety of breeds of dog (Westlake et al., 1995), in Devon cattle (Harper et al., 1988), Rambouillet sheep (Edwards et al., 1994), Nubian goats (Fiske and Storts, 1988), a cynomolgus monkey (Jasty et al., 1984) and mutant mice (Pardo et al., 1994). There are three reports of N C L in cats (Green and Little, 1974; Nakayama et al., 1993; Bildfell et al., 1995), two from Canada (two Siamese cats and a domestic shorthair cat) and one from J a p a n (a domestic cat). This is the first report of N C L in a cat in Europe. The diagnosis was supported by immunohistochemical demonstration of subunit c of mitochondrial ATP synthase. Materials and Methods
Histopathology and Fluorescence Microscopy At necropsy, samples of brain, spinal cord, liver, spleen, kidney, heart and lung were collected and fixed in 7% buffered formalin. The tissues were dehydrated through graded alcohols and, after a xylene step, embedded in paraffin wax and sectioned at 5 gm. The following stains were applied to sections of the central nervous system (CNS): haematoxylin and eosin (HE), periodic acid-Schiff (PAS), Sudan black, luxol fast blue (LFB), Nile blue A and cresyl echt violet. Frozen sections of formalin-fixed material were stained with Sudan III and Sudan black. Unstained frozen sections of formalin-fixed material and unstained dewaxed paraffin-wax sections, as well as HEstained sections, were examined with the fluorescence microscope (excitation filter 450 490 nm, barrier filter 520 rim). Extraneural tissues were stained with HE and examined by light and fluorescence microscopy.
Immunohistochemistry Rabbit antibodies to glial fibrillary acid protein (GFAP) (Sigma, St Louis, USA; dilution 1 in 800) and to subunit c ofmitochondrial ATP synthase (produced according to Palmer et al., 1995; dilution 1 in 1000) were used. Dewaxed sections were incubated with 10% normal goat serum (NGS) for 30min to reduce background staining. Endogenous peroxidase activity was blocked by incubation with 2% H202. The sections were incubated for 16 h at 4~ the primary antibodies being diluted in phosphate-buffered saline (PBS) solution containing NGS 1%. Subsequent steps were made with the Vectastain| ABC-kit (Vector Laboratories, Burlingame, CA, USA) according to the manufacturer's instructions. Brain material from a sheep with NCL served as a positive control for staining of subunit c, and normal cat brain as a negative control.
Electron Microscopy (EM) Formalin-fixed cubes (1 mm 3) from the medulla oblongata were washed for 24 h in PBS, postfixed with osmium tetroxide and embedded in Epon according to standard techniques. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with a Philips 400 T transmission electron microscope. Results
Clinical History A 9-month-old neutered male cat (European domestic shorthair) had a 2week history of uncoordinated gait and episodes of myoclonus and seizures.
N e u r o n a l G e r o i d - l i p o f u s c i n o s i s in a Gat
19
Clinical examination revealed generalized ataxia and strongly reduced postural reactions ("wheelbarrowing" and extensor postural thrust reactions). Spinal reflexes and radiographs were normal. Serological examinations for toxoplasmosis, feline infectious peritonitis, and feline immunodeficiency virus infection produced negative results. The cat was feline leukaemia virusnegative. Treatment for 2 weeks with prednisolone, vitamin-B complex and phenobarbital failed to bring about a clinical improvement, and the animal was unable to recognize food and had severely reduced vision with normal pupillary reflex. At the owner's request the cat was humanely destroyed.
Macroscopical Findings, Histopathology and FluorescenceMicroscopy Marked atrophy of the brain, particularly of the cerebral hemispheres, and a moderate enlargement of the lateral ventricles were noted (Fig. 1). Histologically, an increased cellularity of the cerebral grey matter due to an increased number of glial cells in association with reduced numbers of neurons was seen. The subpial region of the cerebrum showed spongy degeneration and complete loss of neurons. In the cerebellum there was widespread loss of Purkinje cells, which were largely replaced by glial cells located between the molecular and granular layers (Fig. 2). Neurons in all regions of the brain contained single or multiple cytoplasmic inclusions, which were transparent with cresyl echt violet stain, and either transparent or eosinophilic with HE stain (Fig. 3A). Large quantities of storage material were located in the brain-stem nuclei and grey matter of the spinal cord. Some neurons containing large quantities of storage material had undergone lytic necrosis. Storage material stained black with Sudan black in both paraffin-wax and frozen sections. With Nile blue A and LFB, it stained deep .blue (Fig. 3B) and part of the material was weakly pink with PAS. Illumination by ultraviolet light showed bright yellow autofluorescence of unstained frozen and paraffin-wax sections and of HE-stained paraffin-wax sections (Fig. 3C). None of the extraneural tissues examined showed any detectable accumulation of autofluorescent storage material.
Immunohistochemistry GFAP-immunohistochemistry showed marked proliferation of reactive astrocytes in the cerebral hemispheres (Fig. 4) and the granular and Purkinje cell layers of the cerebellum. Subunit c was demonstrated in the cytoplasm of many neurons (Fig. 5) and glial cells. Specific staining was particularly intense in thalamic nuclei, vestibular nuclei and Purkinje cells. In other regions of the brain and in the spinal cord the staining reaction was comparatively weak.
Electron Microscopy (EM) Ultrastructural examination revealed many secondary lysosomes in the cytoplasm of neurons, containing multilamellar arrays consistent with curvilinear
20
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H. Weissenb6ck and C. R6ssel
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3C
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Fig. 1. Transverse section of the atrophic brain of the cat with neuronal ceroid-lipofuscinosis (right), compared with that of a brain from an age-matched cat without neurological disease (left). Fig. 2. Cerebellum with widespread loss of Purkinje cells. The only remaining Purkinje cell is marked by an arrow. HE. x 200. Fig. 3. (A) Neurons with cytoplasmic storage of granular, slightly eosinophilic material (arrows) in the mesencephalon. HE. x 500. (B) Neuronal storage material is stained deep blue with LFB. x 500. (C) Bright yellow autofluorescence of the cytoplasmic storage material. HE, fluorescence micrograph (excitation filter 450 490 nm, barrier filter 520 nm). x 350.
bodies or fingerprint structures. The curvilinear bodies were composed of several (average three) parallel lamellar profiles with a periodicity of 6 nm (Fig. 6A). In some of the bodies the lamellae numbered as many as 10, with an underlay of medium electron-dense homogeneous material. The fingerprint structures showed medium electron density in low magnification. A homogeneous, electron-dense matrix was interspersed with multiple parallel lamellar profiles (periodicity 6 nm) arranged in a fingerprint-pattern (Fig. 6B).
Neuronal
Fig. 4.
Fig. 5.
Geroid-lipofuseinosis
in a Cat
21
(A) Increased number of astrocytes in the cerebral cortex of the cat with neuronal ceroidlipofuscinosis, and (B) normal number of astrocytes in the cerebral cortex of a cat without neurological disease. GFAP-immunohistochemistry. x 85. Subunit c-immunoreactivity of the" cytoplasmic storage material in Purkinje cells and (inset) a neuron from a thalamic nucleus. Immunohistochemistry for subunit c of mitochondrial ATP synthase, x 360. Inset: differential interference contrast, x 500.
22
Fig. 6.
H . Weissenb6ck and G. R 6 s s e l
Curvilinear (A) and fingerprint (B) patterns of the storage material. EM. x 66 000 (A). x 108 000
(B). Discussion
The present case resembled in many ways cases of NCL already reported in man and animals. Thus, the clinical signs (myoclonus, seizures, reduced postural reactions and loss of vision) resemble those reported in man and in the English setter. In sheep, blindness is the major clinical sign, and seizures arid myoclonus have not been reported. In the three previous reports of feline cases, the animals were aged 7-22 months at the onset of disease. Our cat was of comparable age (9 months). The clinical presentations, however, differed. Nakayama et al. (1993) described only shivering and difficulty in walking. Green and Little (1974) and Bildfell et al. (1995) reported convulsions and hyperaesthesia, but no gait abnormalities. Blindness was found only by Bildfell et al. (1995). The major clinical signs in our case were generalized ataxia, reduced postural reactions, seizures, myoclonus and reduced vision. Brain atrophy, as demonstrated by Jolly et al. (1990) in sheep, was also present in our case and in the cat described by Nakayama et al. (1993). The main histological features in the present case (loss of neurons, gliosis and granular neuronal cytoplasmic inclusions) accorded with those of previous reports of NCL in various species. The histochemical staining properties and autofluorescence of the inclusions were indicative of NCL. In most forms of human NCL, the inclusions have a characteristic ultrastructure (curvilinear and fingerprint bodies), also noted in our case. In the cat described here, as in the feline cases of Green and Little (1974) and Bildfell et al. (1995), autofluorescent storage material was not found outside the CNS. Nakayama et al. (1993), however, found storage material in the
Neuronal Ceroid-lipofuscinosis in a Cat
23
mononuclear phagocyte system of the liver, spleen and lymph nodes, in addition to the CNS. Recently, more than half of the storage material in ovine NCL was found to be composed of a hydrophobic protein, the subunit c of mitochondrial ATP synthase (Jolly et al., 1990; Palmer et al., 1992). This protein was also found in several human forms of NCL (Westlake et al., 1995), some canine forms (Westlake et al., 1995), a bovine form (Martinus et al., 1991) and in a "mouse model" (Pardo et al., 1994). It is generally accepted that lysosomal accumulation of subunit c is a cardinal feature in the pathogenesis of NCL. The exact biochemical mechanism leading to the storage is still unknown. A mitochondrial rather than a lysosomal defect may result in accumulation of complexed material that cannot be adequately catabolized (Jolly, 1995). Katz and Siakotos (1995) demonstrated that a specific methylation of one of the lysine residues of subunit c plays an important role in its lysosomal storage. In none of the previously reported feline cases has demonstration ofsubunit c been attempted. Immunohistochemical labelling gave a positive result in the cat described here, which therefore shared an important feature seen in human late infantile NCL and juvenile NCL and in some well defined NCLs in animals. Acknowledgments
The authors thank S. L. Bayliss, Massey University, New Zealand and D. N. Palmer, Lincoln University, New Zealand, for kindly providing antiserum to subunit c and positive control tissue, and for valuable advice. We are grateful to I. Friedl, K. Kain, E. Fitscha and K. Fragner for skilful technical assistance and to K. Bittermann for excellent photographic work. References
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I Received,November29th, 1996] Accepted,March 13th, 1997 J