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Familial motor neuron disease with Lewy body-like inclusions in the substantia nigra, the subthalamic nucleus, and the globus pallidus
Journal of the Neurological Sciences, 108 (1992) 18-23 © 1992 El,,evier Science Publishers B.V. All rights reserved 0022-510X/92/$05.00
18
JNS 03702
Familial motor neuron disease with Lewy body-like inclusions in the substantia nigra, the subthalamic nucleus, and the globus pallidus C h r i s t i n e T r a n c h a n t I, M a r i e - H 6 1 ~ n e D u g a y 1, M i c h e l M o h r and Jean-Marie Warter 1
2, P h i l i p p e
Wasser 1
i Service de lfleurologie li, C.H.U., Strasbourg (France) and 2 Institut d'Anatomie Pathologique, Facultd de Mddecine, Strasbourg (France)
(Received 24 June, 1991) (Revised, received 10 September, 1991) (Accepted 17 September, 1991)
Key words: Motor neuron disease; Luysopallidonigral atrophy; Lewy body-like inclusion Summary In a familial ease of motor neuron disease (MND), 2 unusual features were noted in the necropsy. The first was a pallidoluysonigrai degeneration, observed in only 4 other cases of MND and which was here asymptomatic. The second was the presence in degenerated spinal cord anterior horns and in degenerated basal ganglia of neuronal Lewy body-like inclusions stained by antibodies against ubiquitine.
Introduction
Motor neuron diseases (MND) are usually characterized by an isolated degeneration of peripheral, and sometimes central, motor neurons. However, the degenerative lesions can extend to other parts of the spinal cord or to the basal ganglia (Brownell et al. 1970), and intracellular inclusions are sometimes present (Murayama et al. 1990). We describe a familial form of lower motor neuron disease notable for an asymptomatic pallidoluyso-nigral degeneration and, in all degenerated structures, numerous intraneuronal inclusions resembling Lewy bodies.
Observation A 48-year-old man, without prior illness, was admitted in March 1988 for weakness of the lower limbs, associated with cramps of the right leg, which had started 3 weeks before. Examination showed a proximal amyotrophy of both lower limbs associated with fasciculations of the right quadriceps. Muscle stretch reflexes were present; there was no Babinski sign. The neurological examination was otherwise normal.
Correspondenceto: C. Tranchant, Service de Neurologie 11, C.H.U, 1 place de I'H6pital, 67091 Strasbourg Cedex, France. Tel.: (33)88.16.17.18, ext. 63680; Fax: (33)88.16.12.82.
Metabolic tests were normal. The CSF contained 0.77 g/! protein. The activity of hexoaminidase A was normal. The electromyogram showed signs of distal and proximal neurogenic atrophy of the 4 limbs; sensory and motor conduction velocities were normal. The symptoms worsened rapidly: 8 months later the patient was bedridden. Two months after that, the motor deficit
III 7
I
2
3
4
5
B
? 8
9
10
8
I1 12 13
Fig. 1. Genealogical tree. r~, man; o, woman; / , dead; *, age at death
19 attained the upper limbs, then the respiratory muscles, and death ensued 11 months after the first symptoms. At no point were there a pyramidal syndrome, extrapyramidal signs, abnormal movements, sensory signs, or abnormal cognitive functions. In his family, 4 members over 2 generations have died of flaccid quadriplegia with involvement of the respiratory musculature (Fig. 1).
Materials and methods
Neuropathological examinations Histopathological examination was performed on paraffin sections of blocks taken from various parts of the brain and various levels of the spinal cord, stained with hematoxylin and eosin (H and E). Some sections were also stained with cresyl violet, Nissl technique, P.A.S. and Bodian silver impregnation. Immunocytochemistry was performed on 5-/.tm paraffin sections cut from blocks of spinal cord, of mesencephalon, of thalamic and subthalamic areas and of the lenticular nucleus. The primary antibodies used were a monoclonal mouse anti-neurofilament protein (NF) reacting with the 200-kDa and the 70-kDa subunits of NF (Dakopart), and a monoclonal anti-ubiquitin antibody (DF 2) reacting with free or conjugated forms of ubiquitin. These primary antibodies were diluted with a concentrated solution (10 x ) of proteins (immunized sheep serum + bovine albumin) (Biolyon) as follows: monoclonal antiNF 1:100 and monoclonal DF 2 1:25. The sections were deparaffinized and incubated with the antibodies overnight at room temperature and then visualized by the diaminobenzidine (DAB) reaction with hydrogen peroxyde (universal kit, Biolyon). The immunostained sections were counterstained with hematoxylin.
Results
Cerebral hemispheres, cerebellum, brainstem, spinal cord and spinal nerves were grossly unremarkable. On histological examination, there was a marked neuronal loss and a proportional astrocytic gliosis which affected symetrically the anterior horns of the spinal cord, particularly in the cervical and lumbar enlargements. The cytoplasmic body of the remaining neurons was either normal or chromatolytic. Some neurons showed eosinophilic degeneration. Relatively numerous axonal spheroids were also observed. However, the most remarkable changes were eosinophilic hyaline inclusions that more or less resembled Lewy bodies, in other words Lewy body-like inclusions (LI). These were found in the cytoplasm of many normal or abnormal neurons of the anterior horn (Fig. 2A), they were mostly single,
but sometimes multiple. They were concentric, oval or rod-shaped. Some of them were surrounded by a thin basophilic halo. The L1 were bluish with Nissl's stain, but not with P.A.S. and were not argentophilic. No Bunina bodies were seen. Intermediolateral columns and white matter tracts, particularly in the areas of the cortico-spinal tracts, were normal. In the brainstem, Ll were also found, though less numerous, in the nucleus of the facial nerve, in the nucleus of the oculomotor nerve and in the nucleus ambiguus and the motor nucleus of the trigeminal nerve, where there were also neuronal changes identical to those observed in the anterior horns of the spinal cord. Lastly, eosinophilic inclusions were found in the cytoplasm of some neurons of the brainstem reticular formation. The dorsal vagal nuclei, the hypoglossal nuclei and the locus coeruleus were normal. Bilateral degenerative changes were observed in the substantia nigra, particularly in the zona compacta where groups of pigmented cells had disappeared. There was an associated astrocytic gliosis and melanic pigment was free or intraphagocytic. Several pigmented cells contained intracytoplasmic eosinophilic inclusions, surrounded by a thin clear halo, which were quite similar to Lewy bodies (Fig. 2B). In the diencephalon, there were also degenerative changes affecting the subthalamic nuclei, particularly in their lateral part and the globus pallidus: neuronal loss, an astrocytic gliosis and fairly numerous neuronal LI were encountered (Fig. 2C, D). The other basal ganglia (putamen, caudate, thalamus and nucleus basalis) were normal. The cerebral cortex and white matter were unaffected. The immunocytochemical studies led to the following results (Fig. 3). In the anterior horn of the spinal cord and in the medulla oblongata, only a few inclusions were stained with DF 2. All were stained in their peripheral part, and generally the reaction extended to the surrounding cytoplasm. Furthermore, many tiny spots reactive with DF 2 were seen in the neuropil. Several Ll showed a moderate reaction of their periphery with the antibody raised against NF. Some motor neuron cytoplasmic bodies and some axonal dilatations were also stained with anti-NF antibody. In the substantia nigra, no positive reaction was obtained in U with DF 2, but many perikarya of pigmented neurons without Ll were diffusely stained with DF 2 and numerous tiny spots in the neuropil were also recognized by this antibody. Two LI had their periphery weakly stained with anti-NF antibody. In the subthalamic nucleus, a single U showed, like the previous ones, a staining of its halo with DF 2. Several neuronal perikarya, apparently without inclusions, were also diffusely stained. No staining was obtained with anti-NF antibody. In the globus pallidus, the peripheral halo of a number of LI was stained with DF 2. The cytoplasm
20
Fig. 2. A: an eosinophilic hyaline inclusion in the soma of a cervical anterior horn cell. B, C, D: eosinophilic Lewy body-like inclusions with surrounding halo in the cytoplasm of neurons; B: pigment neuron of substantia nigra; C: subthalamic nucleus; D: globus pallidus (H and E, x 1000).
of a number of other neurons, undergoing degeneration, was diffusely stained. Numerous tiny DF2-positive spots were also observed in the neuropil. The anti-NF antibody stained the halo of several LI.
Discussion The reality of familial forms of MND has been recognized since the work of Kurland and Mulder (1955). Neither their clinical nor their pathological characteristics differ from those of sporadic MND. In a
given family, different clinical aspects can occur (Alajouanine and Nick 1959), and on histologic examination the degeneration of lower motor neuron or of associated lower and upper motor neuron can be seen to extend to other CNS structures (Lawyer and Netsky 1953, Castaigne et al. 1972; Horton et al. 1976, Williams et al. 1990). These associated lesions are perhaps more frequent in familial forms but seem to be identical in the two instances. The most frequently associated lesions are those of the posterior columns, the spinocerebellar tracts and the Clarke columns, which are usually asymptomatic (Castaigne et al. 1972). Degeneration of
21 the basal ganglia is rarer, but not exceptional. Isolated degeneration of the subthalamic nucleus, the striatum, the substantia nigra or the thalamus was observed in 19 of 36 typical amyotrophic lateral sclerosis (ALS) in the series of Brownell et al. (1970). The finding of diffuse pallido-luyso-nigral degeneration, however, is exceptional. Serratrice et al. (1983) reported the case of a woman 54 years o f age who presented a sporadic lower MND that developed over an 1 l-year period, together with a parkinsonian syndrome; autopsy revealed a neuronal loss in the anterior horns of the spinal cord, the
pallidum and the substantia nigra. Gray et al. (1981, 1985) described movement disorders associated with ALS in 2 women in their thirties with familial antecedents of Parkinson's disease. The histopathological study showed cellular depopulation in the anterior horns, atrophy of the corticospinal tracts in the 2nd case, and luyso-pallido-nigral atrophy in both. The only case of asymptomatic pallido-luyso-nigral atrophy associated with a familial MND, outside the case described here, is that of Moya et al. (1969). The patient was a 20-year-old man; autopsy also revealed neuronal loss in
Fig. 3. lmmunocytochemicaistudy of the Lewybody-likeinclusions.A: lumbar anterior horn neuron. Three inclusionsshowing a ring-likepattern of immunostainingwith DF 2. B: another hyaline inclusion in the soma of a lumbar anterior horn neuron showing the same immunostaining pattern with DF2. C: ring-likepattern of NF immunostainingof a hyalin inclusion in the soma of a lumbar anterior horn cell. D: ring-likepattern of NF immunostainingof a hyalin inclusion in a neuron of the nucleus ambiguus(× 1000).
22 the anterior horns and atrophy of the posterior columns. In none of these 4 cases were there special intracytoplasmic inclusions in the atrophied zones as was found in our case. Different types of intracytoplasmic inclusions can be seen in motoneurons of MND patients (Murayama et al. 1990): Bunina bodies (Bunina 1962), basophilic inclusions (Nelson and Prensky 1972), and ILl are the most frequent, but they are non-specific, their presence variable, and their significance poorly understood. LI, like those in our case, were first described in MND by Hirano et al. (1967). They reported 4 patients of the same family presenting a clinical picture of ALS, with intracytoplasmic inclusions localized to the remaining neurons of the anterior horn and the motor nuclei of the medulla oblongata. These inclusions were eosinophilic, formed of a core surrounded by a halo and resembled Lewy's bodies. Thereafter, other authors described inclusions of similar morphology and topographical distribution in several cases of familial MND (Metcalf and Hirano 1971; Takahashi et al. 1972; Kato et al. 1987) and also in cases of sporadic MND (Castaigne et al. 1970; Kato et al. 1988; Kusaka et al. 1988; Sasaki et al. 1989). There seems to be no correlation between the occurrence of these inclusions and the clinical type of MND. Later ultrastructural studies (Castaigne et al. 1970; Takahashi et al. 1972; Goldman and Yen 1986; Kato et al. 1987, 1988; Kusaka et al. 1988; Sasaki et al. 1989) showed that these inclusions, like the typical Lewy body, were constituted of filaments (7 to 20 # m diameter) associated with dense granules and membrane material (Goldman and Yen 1986). Orientation of these filaments and discrimination between halo and core were less precise in MND LI. In immunocytochemical studies (Kato et al. 1989; Murayama et al. 1989, 1990), these LI are, like typical Lewy bodies, stained by antibodies against ubiquitin, but they are not always stained by antibodies against NF. In our case, anterior horn and globus pallidus LI showed a peripheral staining with DF 2, whereas substantia nigra LI did not fix this antibody, and all the LI, in spite of the subthalamic ones, were stained with antibodies against NF. These different patterns of staining could reflect different steps of LI evolution, but could also be explained by technical difficulties in immunocytochemical studies. Since ubiquitine participates in non-lysosomal proteolysis of altered or abnormal proteins (Rechsteiner 1987), L1 may in part be constituted of proteins destined for degradation. LI accumulation in areas of cell degeneration could be evidence of an overwhelmed normal response of the degenerating cell ridding itself of altered proteins (Lowe et al. 1990), or of an abnormal response due to a disturbance of the degradation process.
The presence of these inclusions in about 10% of motorneuron disorders could suggest that the forms with intracytoplasmic inclusions may depend on an etiopathogenic mechanism different from that of other varieties of MND. Acknowledgments
We thank Dr H. Mori and Dr A. Delacourte for helpful advice and the antibodies gift, and Dr F. Gray for helpful advice.
References Alajouanine, T. and J. Nick (1959) Sur trois cas familiaux de scl~rose lat~rale amyotrophique (forme commune; forme bulbaire/l ~volution aigu~; forme ~. type de poliomy~lite ant~rieure chronique) survonue dans la m~me fratrie. Rev. Neurol. (Paris), 100: 490-492. Brownell, B., D.R. Oppenheimer and J.T. Hughes (1970) The central nervous system in motor neurone disease. J. Neurol. Neurosurg. Psychiatry, 33: 338-357. Bunina, T.L. (1962) On intracellular inclusions in familial amyotrophic lateral sclerosis. Korsakov. J. NeuropathoL Psychiatr., 62: 1293-1299. Castaigne, P., J. Cambier, R. Escourolle, P. Brunet and C. Guilleminault (1970) D~g~n~rescence subaigu~ des neurones moteurs p~riph~riques avec inclusions neuronales et lesions des cordons post~rieurs. Les limites de la sel~rose iat&ale amyotrophique. Rev. Neurol. (Paris), 122: 313-320. Castaigne, P., F. Lhermitte, J. Cambier, R. Escourolle and P. Le Bigot (1972) Etude neuropathologique de 61 observations de scl~rose fat&ale amyotrophique, Rev. Neurol. (Paris), 127: 401414. Goldman, ,I.E. and S.H. Yen (1986) Cytoskeletal protein abnormalities in neurodegenerative diseases. Ann. Neurol., 19: 209-223. Gray, F., C. De Baecque, M. Serdaru and R. Escourolle (1981) Pallido-luyso-nigral atrophy and amyotrophic lateral sclerosis. Acta Neuropathol., suppl. VII: 348-351, Gray, F., J.F. Eizenbaum, R. Gherardi, J.D. Degos and J. Poirier (1985) Luyso-pallido-nigralatrophy and amyotrophic lateral sclerosis. Acta Neuropathol., 66: 78-82. Hirano, A., L.T. Kurland and G.P. Sayre (1967) Familial amyotrophic lateral sclerosis. Arch. Neurol, 16: 232-243. Horton, W.A., R. Eldridge and J.A. Brody (1976) Familial motor neuron disease. Neurology, 26: 460-465. Kato, T., A. Hirano and L.T. Kurland (1987) Asymmetric involvement of the spinal cord involving both large and small anterior horn cells in a case of familial amyotrophic lateral sclerosis, Clin. Neuropathol., 6: 67-70. Kato, T., T. Katagiri, A. Hirano, H. Sasaki and S. Arai (1988) Sporadic lower motor neuron disease with Lewy body-like inclusions: a new subgroup. Acta Neuropathol., 76: 208-211. Kato, T., T. Katagiri, A. Hirano, T. Kawanami and H. Sasaki (1989) Lewy body-like hyaline inclusions in sporadic motor neuron disease are ubiquitinated. Acta Neuropathol., 77: 391-396. Kurland, L.T. and D.W. Mulder (1955) Epidemiologic investigations of amyotrophic lateral sclerosis. Part I. Neurology, 5: 182-196. Kurland, L.T. and D.W. Mulder (1955) Epidemiologic investigations of amyotrophic lateral sclerosis. Part 1I. Neurology, 5: 249-268. Kusaka, H., T. lmai, S. Hashimoto, T. Yamamoto, K. Maya and M. Yamasaki (1988) Ultrastructural study of chromatolytic neurons in an adult-onset sporadic case of amyotrophic lateral sclerosis. Acta Neuropathol., 75: 523-528. Lawyer, T. and M.G. Netsky (1953) Amyotrophic lateral sclerosis. A clinico-anatomic study of 53 cases. Arch. Neurol. Psychiatry 69: 171-192.
23 Lowe, J., H. McDermott, M. Landon, R.J. Mayer and K.D. Wilkinson (1990) Ubiquitin carboxyl-terminal hydrolase (PGP 9.5) is selectively present in ubiquitinated inclusion bodies characteristic of human neurodegenerative diseases. J. Pathol., 161: 153-160. Metcalf, C.W. and A. Hirano (1971) Amyotrophic lateral sclerosis. Arch. Neurol., 24: 518-523. Mori, H., J. Kondo and Y. lhara (1987) Ubiquitin is a component of paired helical filaments in Alzheimer's disease. Science, 235: 1641-1644. Moya, G., G. Miranda-Nieves and M. Perez Sotelo (1969) Un cas familial d'amyotrophie spinale progressive montrant une atteinte histologique, cliniquement muette, du pallidum, du locus niger, du noyau de Luys et du faisceau de GoU. Acta Ncurol. Belg., 69: 1002-1012. Murayama, S., H. Mori, Y. Ihara, T.W. Bouldin, H. Suzuki and M. Tomonaga (1990) Immunncytochemical and ultrastructural studies of lower motor neurons in amyotrophic lateral sclerosis. Ann. Neurol., 27: 137-148. Murayama, S., Y. Ookawa and H. Mori H (1989) Immunocytochemi-
cal and ultrastructural study of Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis. Acta Neuropathol., 78: 143-152. Nelson, J.S. and A.L. Prensky (1972) Sporadic juvenile amyotrophic lateral sclerosis. Arch. Neurol., 27: 300-306. Rechsteiner, M. (1987) Ubiquitin-mediated pathways for intracellular proteolysis. Ann. Rev. Ceil. Biol., 3: 1-30. Sasaki, S., K. Yamane, H. Sakuma and S. Maruyama (1989) Sporadic motor neuron disease with Lewy body-like hyaline inclusions. Acta Neuropathol., 78: 555-560. Serratrice, G.T., M. Togaz and J.F. Pellissier (1983) Chronic spinal muscular atrophy and pallidonigral degeneration: report of a case. Neurology, 33: 306-310. Takahashi, K., H. lqakamura and E. Okada (1972) Hereditary amyotrophic lateral sclerosis. Arch. Neurol., 27: 292-299. Williams, C, M.A. Kozlowski, D.R. Hinton and C.A. Miller (1990) Degeneration of spinocerebeUar neurons in amyotrophic lateral sclerosis. Ann. Neurol., 27: 215-225.
18
JNS 03702
Familial motor neuron disease with Lewy body-like inclusions in the substantia nigra, the subthalamic nucleus, and the globus pallidus C h r i s t i n e T r a n c h a n t I, M a r i e - H 6 1 ~ n e D u g a y 1, M i c h e l M o h r and Jean-Marie Warter 1
2, P h i l i p p e
Wasser 1
i Service de lfleurologie li, C.H.U., Strasbourg (France) and 2 Institut d'Anatomie Pathologique, Facultd de Mddecine, Strasbourg (France)
(Received 24 June, 1991) (Revised, received 10 September, 1991) (Accepted 17 September, 1991)
Key words: Motor neuron disease; Luysopallidonigral atrophy; Lewy body-like inclusion Summary In a familial ease of motor neuron disease (MND), 2 unusual features were noted in the necropsy. The first was a pallidoluysonigrai degeneration, observed in only 4 other cases of MND and which was here asymptomatic. The second was the presence in degenerated spinal cord anterior horns and in degenerated basal ganglia of neuronal Lewy body-like inclusions stained by antibodies against ubiquitine.
Introduction
Motor neuron diseases (MND) are usually characterized by an isolated degeneration of peripheral, and sometimes central, motor neurons. However, the degenerative lesions can extend to other parts of the spinal cord or to the basal ganglia (Brownell et al. 1970), and intracellular inclusions are sometimes present (Murayama et al. 1990). We describe a familial form of lower motor neuron disease notable for an asymptomatic pallidoluyso-nigral degeneration and, in all degenerated structures, numerous intraneuronal inclusions resembling Lewy bodies.
Observation A 48-year-old man, without prior illness, was admitted in March 1988 for weakness of the lower limbs, associated with cramps of the right leg, which had started 3 weeks before. Examination showed a proximal amyotrophy of both lower limbs associated with fasciculations of the right quadriceps. Muscle stretch reflexes were present; there was no Babinski sign. The neurological examination was otherwise normal.
Correspondenceto: C. Tranchant, Service de Neurologie 11, C.H.U, 1 place de I'H6pital, 67091 Strasbourg Cedex, France. Tel.: (33)88.16.17.18, ext. 63680; Fax: (33)88.16.12.82.
Metabolic tests were normal. The CSF contained 0.77 g/! protein. The activity of hexoaminidase A was normal. The electromyogram showed signs of distal and proximal neurogenic atrophy of the 4 limbs; sensory and motor conduction velocities were normal. The symptoms worsened rapidly: 8 months later the patient was bedridden. Two months after that, the motor deficit
III 7
I
2
3
4
5
B
? 8
9
10
8
I1 12 13
Fig. 1. Genealogical tree. r~, man; o, woman; / , dead; *, age at death
19 attained the upper limbs, then the respiratory muscles, and death ensued 11 months after the first symptoms. At no point were there a pyramidal syndrome, extrapyramidal signs, abnormal movements, sensory signs, or abnormal cognitive functions. In his family, 4 members over 2 generations have died of flaccid quadriplegia with involvement of the respiratory musculature (Fig. 1).
Materials and methods
Neuropathological examinations Histopathological examination was performed on paraffin sections of blocks taken from various parts of the brain and various levels of the spinal cord, stained with hematoxylin and eosin (H and E). Some sections were also stained with cresyl violet, Nissl technique, P.A.S. and Bodian silver impregnation. Immunocytochemistry was performed on 5-/.tm paraffin sections cut from blocks of spinal cord, of mesencephalon, of thalamic and subthalamic areas and of the lenticular nucleus. The primary antibodies used were a monoclonal mouse anti-neurofilament protein (NF) reacting with the 200-kDa and the 70-kDa subunits of NF (Dakopart), and a monoclonal anti-ubiquitin antibody (DF 2) reacting with free or conjugated forms of ubiquitin. These primary antibodies were diluted with a concentrated solution (10 x ) of proteins (immunized sheep serum + bovine albumin) (Biolyon) as follows: monoclonal antiNF 1:100 and monoclonal DF 2 1:25. The sections were deparaffinized and incubated with the antibodies overnight at room temperature and then visualized by the diaminobenzidine (DAB) reaction with hydrogen peroxyde (universal kit, Biolyon). The immunostained sections were counterstained with hematoxylin.
Results
Cerebral hemispheres, cerebellum, brainstem, spinal cord and spinal nerves were grossly unremarkable. On histological examination, there was a marked neuronal loss and a proportional astrocytic gliosis which affected symetrically the anterior horns of the spinal cord, particularly in the cervical and lumbar enlargements. The cytoplasmic body of the remaining neurons was either normal or chromatolytic. Some neurons showed eosinophilic degeneration. Relatively numerous axonal spheroids were also observed. However, the most remarkable changes were eosinophilic hyaline inclusions that more or less resembled Lewy bodies, in other words Lewy body-like inclusions (LI). These were found in the cytoplasm of many normal or abnormal neurons of the anterior horn (Fig. 2A), they were mostly single,
but sometimes multiple. They were concentric, oval or rod-shaped. Some of them were surrounded by a thin basophilic halo. The L1 were bluish with Nissl's stain, but not with P.A.S. and were not argentophilic. No Bunina bodies were seen. Intermediolateral columns and white matter tracts, particularly in the areas of the cortico-spinal tracts, were normal. In the brainstem, Ll were also found, though less numerous, in the nucleus of the facial nerve, in the nucleus of the oculomotor nerve and in the nucleus ambiguus and the motor nucleus of the trigeminal nerve, where there were also neuronal changes identical to those observed in the anterior horns of the spinal cord. Lastly, eosinophilic inclusions were found in the cytoplasm of some neurons of the brainstem reticular formation. The dorsal vagal nuclei, the hypoglossal nuclei and the locus coeruleus were normal. Bilateral degenerative changes were observed in the substantia nigra, particularly in the zona compacta where groups of pigmented cells had disappeared. There was an associated astrocytic gliosis and melanic pigment was free or intraphagocytic. Several pigmented cells contained intracytoplasmic eosinophilic inclusions, surrounded by a thin clear halo, which were quite similar to Lewy bodies (Fig. 2B). In the diencephalon, there were also degenerative changes affecting the subthalamic nuclei, particularly in their lateral part and the globus pallidus: neuronal loss, an astrocytic gliosis and fairly numerous neuronal LI were encountered (Fig. 2C, D). The other basal ganglia (putamen, caudate, thalamus and nucleus basalis) were normal. The cerebral cortex and white matter were unaffected. The immunocytochemical studies led to the following results (Fig. 3). In the anterior horn of the spinal cord and in the medulla oblongata, only a few inclusions were stained with DF 2. All were stained in their peripheral part, and generally the reaction extended to the surrounding cytoplasm. Furthermore, many tiny spots reactive with DF 2 were seen in the neuropil. Several Ll showed a moderate reaction of their periphery with the antibody raised against NF. Some motor neuron cytoplasmic bodies and some axonal dilatations were also stained with anti-NF antibody. In the substantia nigra, no positive reaction was obtained in U with DF 2, but many perikarya of pigmented neurons without Ll were diffusely stained with DF 2 and numerous tiny spots in the neuropil were also recognized by this antibody. Two LI had their periphery weakly stained with anti-NF antibody. In the subthalamic nucleus, a single U showed, like the previous ones, a staining of its halo with DF 2. Several neuronal perikarya, apparently without inclusions, were also diffusely stained. No staining was obtained with anti-NF antibody. In the globus pallidus, the peripheral halo of a number of LI was stained with DF 2. The cytoplasm
20
Fig. 2. A: an eosinophilic hyaline inclusion in the soma of a cervical anterior horn cell. B, C, D: eosinophilic Lewy body-like inclusions with surrounding halo in the cytoplasm of neurons; B: pigment neuron of substantia nigra; C: subthalamic nucleus; D: globus pallidus (H and E, x 1000).
of a number of other neurons, undergoing degeneration, was diffusely stained. Numerous tiny DF2-positive spots were also observed in the neuropil. The anti-NF antibody stained the halo of several LI.
Discussion The reality of familial forms of MND has been recognized since the work of Kurland and Mulder (1955). Neither their clinical nor their pathological characteristics differ from those of sporadic MND. In a
given family, different clinical aspects can occur (Alajouanine and Nick 1959), and on histologic examination the degeneration of lower motor neuron or of associated lower and upper motor neuron can be seen to extend to other CNS structures (Lawyer and Netsky 1953, Castaigne et al. 1972; Horton et al. 1976, Williams et al. 1990). These associated lesions are perhaps more frequent in familial forms but seem to be identical in the two instances. The most frequently associated lesions are those of the posterior columns, the spinocerebellar tracts and the Clarke columns, which are usually asymptomatic (Castaigne et al. 1972). Degeneration of
21 the basal ganglia is rarer, but not exceptional. Isolated degeneration of the subthalamic nucleus, the striatum, the substantia nigra or the thalamus was observed in 19 of 36 typical amyotrophic lateral sclerosis (ALS) in the series of Brownell et al. (1970). The finding of diffuse pallido-luyso-nigral degeneration, however, is exceptional. Serratrice et al. (1983) reported the case of a woman 54 years o f age who presented a sporadic lower MND that developed over an 1 l-year period, together with a parkinsonian syndrome; autopsy revealed a neuronal loss in the anterior horns of the spinal cord, the
pallidum and the substantia nigra. Gray et al. (1981, 1985) described movement disorders associated with ALS in 2 women in their thirties with familial antecedents of Parkinson's disease. The histopathological study showed cellular depopulation in the anterior horns, atrophy of the corticospinal tracts in the 2nd case, and luyso-pallido-nigral atrophy in both. The only case of asymptomatic pallido-luyso-nigral atrophy associated with a familial MND, outside the case described here, is that of Moya et al. (1969). The patient was a 20-year-old man; autopsy also revealed neuronal loss in
Fig. 3. lmmunocytochemicaistudy of the Lewybody-likeinclusions.A: lumbar anterior horn neuron. Three inclusionsshowing a ring-likepattern of immunostainingwith DF 2. B: another hyaline inclusion in the soma of a lumbar anterior horn neuron showing the same immunostaining pattern with DF2. C: ring-likepattern of NF immunostainingof a hyalin inclusion in the soma of a lumbar anterior horn cell. D: ring-likepattern of NF immunostainingof a hyalin inclusion in a neuron of the nucleus ambiguus(× 1000).
22 the anterior horns and atrophy of the posterior columns. In none of these 4 cases were there special intracytoplasmic inclusions in the atrophied zones as was found in our case. Different types of intracytoplasmic inclusions can be seen in motoneurons of MND patients (Murayama et al. 1990): Bunina bodies (Bunina 1962), basophilic inclusions (Nelson and Prensky 1972), and ILl are the most frequent, but they are non-specific, their presence variable, and their significance poorly understood. LI, like those in our case, were first described in MND by Hirano et al. (1967). They reported 4 patients of the same family presenting a clinical picture of ALS, with intracytoplasmic inclusions localized to the remaining neurons of the anterior horn and the motor nuclei of the medulla oblongata. These inclusions were eosinophilic, formed of a core surrounded by a halo and resembled Lewy's bodies. Thereafter, other authors described inclusions of similar morphology and topographical distribution in several cases of familial MND (Metcalf and Hirano 1971; Takahashi et al. 1972; Kato et al. 1987) and also in cases of sporadic MND (Castaigne et al. 1970; Kato et al. 1988; Kusaka et al. 1988; Sasaki et al. 1989). There seems to be no correlation between the occurrence of these inclusions and the clinical type of MND. Later ultrastructural studies (Castaigne et al. 1970; Takahashi et al. 1972; Goldman and Yen 1986; Kato et al. 1987, 1988; Kusaka et al. 1988; Sasaki et al. 1989) showed that these inclusions, like the typical Lewy body, were constituted of filaments (7 to 20 # m diameter) associated with dense granules and membrane material (Goldman and Yen 1986). Orientation of these filaments and discrimination between halo and core were less precise in MND LI. In immunocytochemical studies (Kato et al. 1989; Murayama et al. 1989, 1990), these LI are, like typical Lewy bodies, stained by antibodies against ubiquitin, but they are not always stained by antibodies against NF. In our case, anterior horn and globus pallidus LI showed a peripheral staining with DF 2, whereas substantia nigra LI did not fix this antibody, and all the LI, in spite of the subthalamic ones, were stained with antibodies against NF. These different patterns of staining could reflect different steps of LI evolution, but could also be explained by technical difficulties in immunocytochemical studies. Since ubiquitine participates in non-lysosomal proteolysis of altered or abnormal proteins (Rechsteiner 1987), L1 may in part be constituted of proteins destined for degradation. LI accumulation in areas of cell degeneration could be evidence of an overwhelmed normal response of the degenerating cell ridding itself of altered proteins (Lowe et al. 1990), or of an abnormal response due to a disturbance of the degradation process.
The presence of these inclusions in about 10% of motorneuron disorders could suggest that the forms with intracytoplasmic inclusions may depend on an etiopathogenic mechanism different from that of other varieties of MND. Acknowledgments
We thank Dr H. Mori and Dr A. Delacourte for helpful advice and the antibodies gift, and Dr F. Gray for helpful advice.
References Alajouanine, T. and J. Nick (1959) Sur trois cas familiaux de scl~rose lat~rale amyotrophique (forme commune; forme bulbaire/l ~volution aigu~; forme ~. type de poliomy~lite ant~rieure chronique) survonue dans la m~me fratrie. Rev. Neurol. (Paris), 100: 490-492. Brownell, B., D.R. Oppenheimer and J.T. Hughes (1970) The central nervous system in motor neurone disease. J. Neurol. Neurosurg. Psychiatry, 33: 338-357. Bunina, T.L. (1962) On intracellular inclusions in familial amyotrophic lateral sclerosis. Korsakov. J. NeuropathoL Psychiatr., 62: 1293-1299. Castaigne, P., J. Cambier, R. Escourolle, P. Brunet and C. Guilleminault (1970) D~g~n~rescence subaigu~ des neurones moteurs p~riph~riques avec inclusions neuronales et lesions des cordons post~rieurs. Les limites de la sel~rose iat&ale amyotrophique. Rev. Neurol. (Paris), 122: 313-320. Castaigne, P., F. Lhermitte, J. Cambier, R. Escourolle and P. Le Bigot (1972) Etude neuropathologique de 61 observations de scl~rose fat&ale amyotrophique, Rev. Neurol. (Paris), 127: 401414. Goldman, ,I.E. and S.H. Yen (1986) Cytoskeletal protein abnormalities in neurodegenerative diseases. Ann. Neurol., 19: 209-223. Gray, F., C. De Baecque, M. Serdaru and R. Escourolle (1981) Pallido-luyso-nigral atrophy and amyotrophic lateral sclerosis. Acta Neuropathol., suppl. VII: 348-351, Gray, F., J.F. Eizenbaum, R. Gherardi, J.D. Degos and J. Poirier (1985) Luyso-pallido-nigralatrophy and amyotrophic lateral sclerosis. Acta Neuropathol., 66: 78-82. Hirano, A., L.T. Kurland and G.P. Sayre (1967) Familial amyotrophic lateral sclerosis. Arch. Neurol, 16: 232-243. Horton, W.A., R. Eldridge and J.A. Brody (1976) Familial motor neuron disease. Neurology, 26: 460-465. Kato, T., A. Hirano and L.T. Kurland (1987) Asymmetric involvement of the spinal cord involving both large and small anterior horn cells in a case of familial amyotrophic lateral sclerosis, Clin. Neuropathol., 6: 67-70. Kato, T., T. Katagiri, A. Hirano, H. Sasaki and S. Arai (1988) Sporadic lower motor neuron disease with Lewy body-like inclusions: a new subgroup. Acta Neuropathol., 76: 208-211. Kato, T., T. Katagiri, A. Hirano, T. Kawanami and H. Sasaki (1989) Lewy body-like hyaline inclusions in sporadic motor neuron disease are ubiquitinated. Acta Neuropathol., 77: 391-396. Kurland, L.T. and D.W. Mulder (1955) Epidemiologic investigations of amyotrophic lateral sclerosis. Part I. Neurology, 5: 182-196. Kurland, L.T. and D.W. Mulder (1955) Epidemiologic investigations of amyotrophic lateral sclerosis. Part 1I. Neurology, 5: 249-268. Kusaka, H., T. lmai, S. Hashimoto, T. Yamamoto, K. Maya and M. Yamasaki (1988) Ultrastructural study of chromatolytic neurons in an adult-onset sporadic case of amyotrophic lateral sclerosis. Acta Neuropathol., 75: 523-528. Lawyer, T. and M.G. Netsky (1953) Amyotrophic lateral sclerosis. A clinico-anatomic study of 53 cases. Arch. Neurol. Psychiatry 69: 171-192.
23 Lowe, J., H. McDermott, M. Landon, R.J. Mayer and K.D. Wilkinson (1990) Ubiquitin carboxyl-terminal hydrolase (PGP 9.5) is selectively present in ubiquitinated inclusion bodies characteristic of human neurodegenerative diseases. J. Pathol., 161: 153-160. Metcalf, C.W. and A. Hirano (1971) Amyotrophic lateral sclerosis. Arch. Neurol., 24: 518-523. Mori, H., J. Kondo and Y. lhara (1987) Ubiquitin is a component of paired helical filaments in Alzheimer's disease. Science, 235: 1641-1644. Moya, G., G. Miranda-Nieves and M. Perez Sotelo (1969) Un cas familial d'amyotrophie spinale progressive montrant une atteinte histologique, cliniquement muette, du pallidum, du locus niger, du noyau de Luys et du faisceau de GoU. Acta Ncurol. Belg., 69: 1002-1012. Murayama, S., H. Mori, Y. Ihara, T.W. Bouldin, H. Suzuki and M. Tomonaga (1990) Immunncytochemical and ultrastructural studies of lower motor neurons in amyotrophic lateral sclerosis. Ann. Neurol., 27: 137-148. Murayama, S., Y. Ookawa and H. Mori H (1989) Immunocytochemi-
cal and ultrastructural study of Lewy body-like hyaline inclusions in familial amyotrophic lateral sclerosis. Acta Neuropathol., 78: 143-152. Nelson, J.S. and A.L. Prensky (1972) Sporadic juvenile amyotrophic lateral sclerosis. Arch. Neurol., 27: 300-306. Rechsteiner, M. (1987) Ubiquitin-mediated pathways for intracellular proteolysis. Ann. Rev. Ceil. Biol., 3: 1-30. Sasaki, S., K. Yamane, H. Sakuma and S. Maruyama (1989) Sporadic motor neuron disease with Lewy body-like hyaline inclusions. Acta Neuropathol., 78: 555-560. Serratrice, G.T., M. Togaz and J.F. Pellissier (1983) Chronic spinal muscular atrophy and pallidonigral degeneration: report of a case. Neurology, 33: 306-310. Takahashi, K., H. lqakamura and E. Okada (1972) Hereditary amyotrophic lateral sclerosis. Arch. Neurol., 27: 292-299. Williams, C, M.A. Kozlowski, D.R. Hinton and C.A. Miller (1990) Degeneration of spinocerebeUar neurons in amyotrophic lateral sclerosis. Ann. Neurol., 27: 215-225.