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Ultrastructural study of Bunina bodies in the anterior horn neurons of patients with amyotrophic lateral sclerosis
117
Neuroscience Letters, 154 (1993) 117-120 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
NSL 09496
Ultrastructural study of Bunina bodies in the anterior horn neurons of patients with amyotrophic lateral sclerosis Shoichi Sasaki and Shoichi M a r u y a m a Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo (Japan) (Received 2 November 1992; Revised version received 10 February 1993; Accepted 10 February 1993)
Key words. Amyotrophic lateral sclerosis; Bunina body; Electron microscopy We ultrastructurally investigated Bunina bodies (BB) in the anterior horn neurons of 20 amyotrophic lateral sclerosis patients with BB. As for novel findings, filaments thicker than neurofilaments were not uncommonly observed inside the BB. They were occasionally observed around the periphery of the BB. Some of them were composed of bundles of filaments which appeared constricted at 40-50 nm intervals and were 20-25 nm in maximum width. Others consisted of bundles of unconstricted filaments measuring about 20-25 nm in diameter. The BB occasionally contained bundles of filaments of about 20 nm in diameter that closely resembled those found in ubiquitin-positive skein-like inclusions. It seems that some molecular disturbances such as ubiquitin play a role in the formation of the constricted and unconstricted filaments. Probably cytoskeletal or non-cytoskeletal proteins in anterior horn cells are damaged and accumulate to form aggregation of the filaments associated with BB.
Small eosinophilic Bunina bodies (BB) are generally considered to be a specific pathologic hallmark of amyotrophic lateral sclerosis (ALS), since they are an almost constant accompaniment of this disease and appear to be exclusive to it [1, 3]. Despite several ultrastructural investigations [2, 9, 10, 14], the precise nature and origin of BB still remain unclear. In this study, we investigated BB in ALS patients by, electron microscopy to shed some more light on their uitrastructure. The spinal cords of 20 ALS patients with BB were investigated. The patients ranged from 47 to 83 years of age (average: 65.0 years). Autopsy was performed on all patients within 6 h of death. The lumbosacral segments (Ll_ 5 and S 1 2) of the spinal cords were fixed in 2% glutaraldehyde with phosphate buffer (pH 7.40). After fixation, the anterior horn tissue at each level was cut into small pieces, postfixed in 1% osmium tetroxide for several hours, dehydrated, and embedded in epoxy resin. The embedded specimens were then cut into semithin (1 am) sections and stained with Toluidine blue. Appropriate regions were properly trimmed and subsequently cut into serial ultrathin sections, which were stained with uranyl acetate and lead citrate for the electron micro-
Correspondence: Shoichi Sasaki, Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo 162, Japan. Fax: (81) 3 5269 7324.
scopic study. JEOL 100 CX electron microscope was used. As controls, the same regions of 14 patients who died without any known neurological disease were investigated by the same procedure. These controls ranged from 44 to 80 years in age (average: 59.4 years). BB were observed not only in neurons featuring chromatolysis or lipofuscin accumulation, but also in normal-appearing anterior horn neurons. In general, the BB consisted mainly of electron-dense granular or amorphous material that was usually surrounded or invaginated by vesicles and tubular structures (Fig. 1). However, some BB were predominantly composed of vesicles and tubular structures and had less electron-dense material, while others consisted exclusively of a cluster of granules or amorphous material. The BB often contained cytoplasmic organelles such as 10 nm neurofilaments, mitochondria and lipofuscin granules (Fig. 1). Filaments thicker than neurofilaments were not uncommon in the BB, especially in those of an ALS patient with dementia. Some of them were composed of bundles which appeared to bear constrictions at about 40-50 nm intervals and were between 20 and 25 nm in maximum width (Fig. 2). On transverse sections, the constrictions showed a circular profile. Others consisted of bundles of the filaments without constrictions and measured approximately 20-25 nm in diameter (Fig. 3). The constricted and unconstricted filaments were occasionally observed around the periphery of the BB (Fig. 4). The
118 t
~!~!~i~~¸ i i~
Fig. 1. Electron micrograph of a Bunina body consisting of electrondense material containing an accumulation of neurofilaments surrounded by vesicles and tubular structures, xl 9,000
Fig. 2. Filaments thicker than neurofilaments are observed within the Bunina body. The filaments have a twist at 40 50 nm intervals, and their maximum width is 20 25 nm. x38,000
BB occasionally contained bundles o f filaments o f about 20 n m in diameter that closely resembled the filaments o f skein-like inclusions (Fig. 5). A r o u n d the periphery o f the BB were accumulations o f cytoplasmic organelles. such as mitochondria, Nissl granules and lipofuscin granules. These structures were sometimes directly attached to the BB. Occasionally, intermediate filaments were accumulated both a r o u n d and within the BB (Fig. 6). M i t o c h o n d r i a containing irregular deposits o f electron-dense material and paracrystalline arrays resembling H i r a n o bodies or lamellar bodies were only occasionally observed in the vicinity of BB. BB were usually found in the perikarya o f anterior horn neurons and were not u n c o m m o n in the primary dendrites, but were never seen in the axons. BB were only occasionally found in the postsynaptic region. W h e n BB were observed in the prim a r y dendrites, they were frequently sandwiched be-
tween collections o f Nissl substance. A m o n g the controls, three patients rarely showed electron-dense material resembling BB in the perikarya o f anterior horn neurons, but there were no BB observed. Several studies on the ultrastructure o f BB have already been published [2. 9, 10, 14] and the fine structure noted in this study is basically consistent with that already reported. A significant novel finding is that o f 2025 nm filaments within the inclusions. Some o f them appear to have constrictions and closely resemble the paired helical filaments seen in spinal ganglion neurons o f senescent rats [16]. They are also similar to the paired filaments f o u n d in the dendritic terminals o f the cerebral cortex in senescent rhesus m o n k e y s [17], while different from the neurofibrillary tangles seen in Alzheimer's disease (AD) [13]. On the other hand, the unconstricted filaments noted in the current study closely resemble the
Fig. 3. The filaments are unconstricted and approximately 20 25 nm in diameter, x25,600
Fig. 4. Filaments thicker than neurofilaments around the periphery of the Bunina Body. x25,600
119
Fig. 5. A Bunina body containing bundles of filaments (arrows) approximately 20 nm in diameter that closely resemble those seen in skeinlike inclusion, x25,600
so-called curly fibers or threads o f neuronal processes noted in A D [18]. They are different f r o m the 15-nm straight tubules f o u n d in progressive supranuclear palsy (PSP) [14]. Moreover, the present study showed that BB occasionally contained bundles o f filaments closely resembling those c o m p o s i n g ubiquitin-positive skein-like inclusions. In this light, ubiquitin might be possible comp o n e n t o f 20-25 n m filaments. The diversity o f etiology o f the constricted and unconstricted filaments makes it likely that some molecular disturbances such as ubiquitin play a c o m m o n role in the formation o f the filaments. The presence o f the 20-25 n m filaments in both sporadic and demented A L S patients m a y fortify the above assumption, namely, c o m m o n mechanism o f the formation o f the filaments. Lowe et al. [5] and M u r a y a m a et al. [8] suggested a close relationship between BB and ubiquitinreactive inclusions. Lowe et al. [5] reported that BB were always surrounded by large ubiquitin-positive inclusions. M u r a y a m a et al. [8] suggested that bundles o f coated filaments (12 n m in diameter), which p r o b a b l y corresponded to ubiquitin-positive skein-like inclusions [7, 11, 12] and aggregated to form BB-like structures, might be a precursor o f BB. O u r finding o f previously unrecognized 20-25 n m filaments suggests that p r o b a b l y cytoskeletal or non-cytoskeletal proteins in anterior h o r n cells are d a m a g e d and accumulate to form aggregation o f the filaments associated with BB. This work was supported by Research for New D r u g Development in A L S from the Japanese Ministry o f Health and Welfare. We wish to thank Prof. A. H i r a n o (Division o f N e u r o p a t h o l o g y , D e p a r t m e n t o f Pathology, Montefiore Medical Center) for his m a n y valuable suggestions and Dr. H. K u s a k a ( D e p a r t m e n t o f Neurology,
Fig. 6. An accumulation of neurofilaments is observed around and within a Bunina body. x19,000
K i t a n o Hospital and Neurological Center) for giving us a chance to investigate two A L S patients. 1 Bunina, T.L., On intracellular inclusions in familial amyotrophic lateral sclerosis, Korsakov J. Neuropathol. Psychiat., 62 (1962) 1293 1299. 2 Hart, M.N., Cancilla, P.A., Frommes, S. and Hirano, A., Anterior horn cell degeneration and Bunina-type inclusions associated with dementia, Acta Neuropathol. (Berl.), 38 (1977) 225-228. 3 Hirano, A. (Ed.), In: A Guide to Neuropathology, Igaku-shoin, Tokyo, 1981, pp. 162-164. 4 Deleted. 5 Lowe, J., Lennox, G., Jefferson, D., Morrell, K., McQuire, D., Gray, T., Landon, M., Doherty, F.J. and Mayer, R.J., A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localization of ubiquitin, Neurosci. Lett., 94 (1988) 203 210. 6 Deleted. 7 Mizusawa, H., Nakamura, H., Wakayama, I., Yen, S.-H.C. and Hirano, A., Skein-like inclusions in the anterior horn cells in motor neuron disease, J. Neurol. Sci., 105 (1991) 14-21. 8 Murayama, S., Mori, H., lhara, Y., Bouldin, T.W., Suzuki, K. and Tomonaga, M., Immunocytochemical and ultra-structural studies of lower motor neurons in amyotrophic lateral sclerosis, Ann. Neurol., 27 (1990) 137 148. 9 Okamoto, K., Morimatsu, M., Hirai, S., and Ishida, Y., Intracytoplasmic inclusions (Bunina bodies) in amyotrophic lateral sclerosis, Acta Pathol. Jpn., 30 (1980) 591 597. 10 Sasaki, S., Hirano, A., Donnenfeld, H. and Nakano, I., An electron microscopic study of Bunina bodies, Neurol. Med. (Tokyo), 19 (1983) 317-324. 11 Sasaki, S. and Maruyama, S., Immunocytochemical and ultrastructural studies of hyaline inclusions in sporadic motor neuron disease, Acta Neuropathol., 82 (1991) 295-301. 12 Sasaki, S. and Maruyama, S., Ultrastructural study of skein-like inclusions in anterior horn neurons of patients with motor neuron disease, Neurosci. Lett., 147 (1992) 121 124. 13 Terry, R.D., The fine structure of neurofibrillary tangles in Alzheimer's disease, J. Neuropathol. Exp. Neurol., 2 (1963) 629-642.
120 14 Tomonaga, M., Ultrastructure of neurofibrillary tangles in progressive supranuclear palsy, Acta Neuropathol. (Berl.), 37 (1977) 177 181. 15 Tomonaga, M., Saito, M., Yoshimura, M., Shimada, H. and Tohgi, H., Ultrastructure of the Bunina bodies in anterior horn cells of amyotrophic lateral sclerosis, Acta Neuropathol. (Berl.), 42 (1978) 81-86. 16 Van den Bosch de Aguilar, Eh. and Goemaere-Vanneste, J., Paired helical filaments in spinal ganglion neurons of elderly rats, Virchows Arch. Cell Pathol., 47 (1984) 217-222.
17 Wisniewski, H.M., Ghetti, B. and Terry, R.D., Neuritic (senile) plaques and filamentous changes in aged rhesus monkeys, J. Neuropathol. Exp. Neurol., 32 (1973) 566-584. 18 Yamaguchi, H., Nakazato, Y., Shoji, M., Ihara, Y. and Hirai, S., Ultrastructure of the neuropil threads in the Alzheimer brain; their dendritic origin and accumulation in the senile plaques, Acta Neuropathol., 80 (1990) 368 374.
Neuroscience Letters, 154 (1993) 117-120 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/93/$ 06.00
NSL 09496
Ultrastructural study of Bunina bodies in the anterior horn neurons of patients with amyotrophic lateral sclerosis Shoichi Sasaki and Shoichi M a r u y a m a Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo (Japan) (Received 2 November 1992; Revised version received 10 February 1993; Accepted 10 February 1993)
Key words. Amyotrophic lateral sclerosis; Bunina body; Electron microscopy We ultrastructurally investigated Bunina bodies (BB) in the anterior horn neurons of 20 amyotrophic lateral sclerosis patients with BB. As for novel findings, filaments thicker than neurofilaments were not uncommonly observed inside the BB. They were occasionally observed around the periphery of the BB. Some of them were composed of bundles of filaments which appeared constricted at 40-50 nm intervals and were 20-25 nm in maximum width. Others consisted of bundles of unconstricted filaments measuring about 20-25 nm in diameter. The BB occasionally contained bundles of filaments of about 20 nm in diameter that closely resembled those found in ubiquitin-positive skein-like inclusions. It seems that some molecular disturbances such as ubiquitin play a role in the formation of the constricted and unconstricted filaments. Probably cytoskeletal or non-cytoskeletal proteins in anterior horn cells are damaged and accumulate to form aggregation of the filaments associated with BB.
Small eosinophilic Bunina bodies (BB) are generally considered to be a specific pathologic hallmark of amyotrophic lateral sclerosis (ALS), since they are an almost constant accompaniment of this disease and appear to be exclusive to it [1, 3]. Despite several ultrastructural investigations [2, 9, 10, 14], the precise nature and origin of BB still remain unclear. In this study, we investigated BB in ALS patients by, electron microscopy to shed some more light on their uitrastructure. The spinal cords of 20 ALS patients with BB were investigated. The patients ranged from 47 to 83 years of age (average: 65.0 years). Autopsy was performed on all patients within 6 h of death. The lumbosacral segments (Ll_ 5 and S 1 2) of the spinal cords were fixed in 2% glutaraldehyde with phosphate buffer (pH 7.40). After fixation, the anterior horn tissue at each level was cut into small pieces, postfixed in 1% osmium tetroxide for several hours, dehydrated, and embedded in epoxy resin. The embedded specimens were then cut into semithin (1 am) sections and stained with Toluidine blue. Appropriate regions were properly trimmed and subsequently cut into serial ultrathin sections, which were stained with uranyl acetate and lead citrate for the electron micro-
Correspondence: Shoichi Sasaki, Department of Neurology, Neurological Institute, Tokyo Women's Medical College, Tokyo 162, Japan. Fax: (81) 3 5269 7324.
scopic study. JEOL 100 CX electron microscope was used. As controls, the same regions of 14 patients who died without any known neurological disease were investigated by the same procedure. These controls ranged from 44 to 80 years in age (average: 59.4 years). BB were observed not only in neurons featuring chromatolysis or lipofuscin accumulation, but also in normal-appearing anterior horn neurons. In general, the BB consisted mainly of electron-dense granular or amorphous material that was usually surrounded or invaginated by vesicles and tubular structures (Fig. 1). However, some BB were predominantly composed of vesicles and tubular structures and had less electron-dense material, while others consisted exclusively of a cluster of granules or amorphous material. The BB often contained cytoplasmic organelles such as 10 nm neurofilaments, mitochondria and lipofuscin granules (Fig. 1). Filaments thicker than neurofilaments were not uncommon in the BB, especially in those of an ALS patient with dementia. Some of them were composed of bundles which appeared to bear constrictions at about 40-50 nm intervals and were between 20 and 25 nm in maximum width (Fig. 2). On transverse sections, the constrictions showed a circular profile. Others consisted of bundles of the filaments without constrictions and measured approximately 20-25 nm in diameter (Fig. 3). The constricted and unconstricted filaments were occasionally observed around the periphery of the BB (Fig. 4). The
118 t
~!~!~i~~¸ i i~
Fig. 1. Electron micrograph of a Bunina body consisting of electrondense material containing an accumulation of neurofilaments surrounded by vesicles and tubular structures, xl 9,000
Fig. 2. Filaments thicker than neurofilaments are observed within the Bunina body. The filaments have a twist at 40 50 nm intervals, and their maximum width is 20 25 nm. x38,000
BB occasionally contained bundles o f filaments o f about 20 n m in diameter that closely resembled the filaments o f skein-like inclusions (Fig. 5). A r o u n d the periphery o f the BB were accumulations o f cytoplasmic organelles. such as mitochondria, Nissl granules and lipofuscin granules. These structures were sometimes directly attached to the BB. Occasionally, intermediate filaments were accumulated both a r o u n d and within the BB (Fig. 6). M i t o c h o n d r i a containing irregular deposits o f electron-dense material and paracrystalline arrays resembling H i r a n o bodies or lamellar bodies were only occasionally observed in the vicinity of BB. BB were usually found in the perikarya o f anterior horn neurons and were not u n c o m m o n in the primary dendrites, but were never seen in the axons. BB were only occasionally found in the postsynaptic region. W h e n BB were observed in the prim a r y dendrites, they were frequently sandwiched be-
tween collections o f Nissl substance. A m o n g the controls, three patients rarely showed electron-dense material resembling BB in the perikarya o f anterior horn neurons, but there were no BB observed. Several studies on the ultrastructure o f BB have already been published [2. 9, 10, 14] and the fine structure noted in this study is basically consistent with that already reported. A significant novel finding is that o f 2025 nm filaments within the inclusions. Some o f them appear to have constrictions and closely resemble the paired helical filaments seen in spinal ganglion neurons o f senescent rats [16]. They are also similar to the paired filaments f o u n d in the dendritic terminals o f the cerebral cortex in senescent rhesus m o n k e y s [17], while different from the neurofibrillary tangles seen in Alzheimer's disease (AD) [13]. On the other hand, the unconstricted filaments noted in the current study closely resemble the
Fig. 3. The filaments are unconstricted and approximately 20 25 nm in diameter, x25,600
Fig. 4. Filaments thicker than neurofilaments around the periphery of the Bunina Body. x25,600
119
Fig. 5. A Bunina body containing bundles of filaments (arrows) approximately 20 nm in diameter that closely resemble those seen in skeinlike inclusion, x25,600
so-called curly fibers or threads o f neuronal processes noted in A D [18]. They are different f r o m the 15-nm straight tubules f o u n d in progressive supranuclear palsy (PSP) [14]. Moreover, the present study showed that BB occasionally contained bundles o f filaments closely resembling those c o m p o s i n g ubiquitin-positive skein-like inclusions. In this light, ubiquitin might be possible comp o n e n t o f 20-25 n m filaments. The diversity o f etiology o f the constricted and unconstricted filaments makes it likely that some molecular disturbances such as ubiquitin play a c o m m o n role in the formation o f the filaments. The presence o f the 20-25 n m filaments in both sporadic and demented A L S patients m a y fortify the above assumption, namely, c o m m o n mechanism o f the formation o f the filaments. Lowe et al. [5] and M u r a y a m a et al. [8] suggested a close relationship between BB and ubiquitinreactive inclusions. Lowe et al. [5] reported that BB were always surrounded by large ubiquitin-positive inclusions. M u r a y a m a et al. [8] suggested that bundles o f coated filaments (12 n m in diameter), which p r o b a b l y corresponded to ubiquitin-positive skein-like inclusions [7, 11, 12] and aggregated to form BB-like structures, might be a precursor o f BB. O u r finding o f previously unrecognized 20-25 n m filaments suggests that p r o b a b l y cytoskeletal or non-cytoskeletal proteins in anterior h o r n cells are d a m a g e d and accumulate to form aggregation o f the filaments associated with BB. This work was supported by Research for New D r u g Development in A L S from the Japanese Ministry o f Health and Welfare. We wish to thank Prof. A. H i r a n o (Division o f N e u r o p a t h o l o g y , D e p a r t m e n t o f Pathology, Montefiore Medical Center) for his m a n y valuable suggestions and Dr. H. K u s a k a ( D e p a r t m e n t o f Neurology,
Fig. 6. An accumulation of neurofilaments is observed around and within a Bunina body. x19,000
K i t a n o Hospital and Neurological Center) for giving us a chance to investigate two A L S patients. 1 Bunina, T.L., On intracellular inclusions in familial amyotrophic lateral sclerosis, Korsakov J. Neuropathol. Psychiat., 62 (1962) 1293 1299. 2 Hart, M.N., Cancilla, P.A., Frommes, S. and Hirano, A., Anterior horn cell degeneration and Bunina-type inclusions associated with dementia, Acta Neuropathol. (Berl.), 38 (1977) 225-228. 3 Hirano, A. (Ed.), In: A Guide to Neuropathology, Igaku-shoin, Tokyo, 1981, pp. 162-164. 4 Deleted. 5 Lowe, J., Lennox, G., Jefferson, D., Morrell, K., McQuire, D., Gray, T., Landon, M., Doherty, F.J. and Mayer, R.J., A filamentous inclusion body within anterior horn neurones in motor neurone disease defined by immunocytochemical localization of ubiquitin, Neurosci. Lett., 94 (1988) 203 210. 6 Deleted. 7 Mizusawa, H., Nakamura, H., Wakayama, I., Yen, S.-H.C. and Hirano, A., Skein-like inclusions in the anterior horn cells in motor neuron disease, J. Neurol. Sci., 105 (1991) 14-21. 8 Murayama, S., Mori, H., lhara, Y., Bouldin, T.W., Suzuki, K. and Tomonaga, M., Immunocytochemical and ultra-structural studies of lower motor neurons in amyotrophic lateral sclerosis, Ann. Neurol., 27 (1990) 137 148. 9 Okamoto, K., Morimatsu, M., Hirai, S., and Ishida, Y., Intracytoplasmic inclusions (Bunina bodies) in amyotrophic lateral sclerosis, Acta Pathol. Jpn., 30 (1980) 591 597. 10 Sasaki, S., Hirano, A., Donnenfeld, H. and Nakano, I., An electron microscopic study of Bunina bodies, Neurol. Med. (Tokyo), 19 (1983) 317-324. 11 Sasaki, S. and Maruyama, S., Immunocytochemical and ultrastructural studies of hyaline inclusions in sporadic motor neuron disease, Acta Neuropathol., 82 (1991) 295-301. 12 Sasaki, S. and Maruyama, S., Ultrastructural study of skein-like inclusions in anterior horn neurons of patients with motor neuron disease, Neurosci. Lett., 147 (1992) 121 124. 13 Terry, R.D., The fine structure of neurofibrillary tangles in Alzheimer's disease, J. Neuropathol. Exp. Neurol., 2 (1963) 629-642.
120 14 Tomonaga, M., Ultrastructure of neurofibrillary tangles in progressive supranuclear palsy, Acta Neuropathol. (Berl.), 37 (1977) 177 181. 15 Tomonaga, M., Saito, M., Yoshimura, M., Shimada, H. and Tohgi, H., Ultrastructure of the Bunina bodies in anterior horn cells of amyotrophic lateral sclerosis, Acta Neuropathol. (Berl.), 42 (1978) 81-86. 16 Van den Bosch de Aguilar, Eh. and Goemaere-Vanneste, J., Paired helical filaments in spinal ganglion neurons of elderly rats, Virchows Arch. Cell Pathol., 47 (1984) 217-222.
17 Wisniewski, H.M., Ghetti, B. and Terry, R.D., Neuritic (senile) plaques and filamentous changes in aged rhesus monkeys, J. Neuropathol. Exp. Neurol., 32 (1973) 566-584. 18 Yamaguchi, H., Nakazato, Y., Shoji, M., Ihara, Y. and Hirai, S., Ultrastructure of the neuropil threads in the Alzheimer brain; their dendritic origin and accumulation in the senile plaques, Acta Neuropathol., 80 (1990) 368 374.