Acta Neuropathol (1991) 82:340 - 345
Acta Heuropatholog,ca O Springer-Verlag 1991
Reexamination of granulovacuolar degeneration K. Okamoto, S. Hirai, T. Iizuka, T. Yanagisawa, and M. Watanabe Department of Neurology, Gunma University School of Medicine, Maebashi, Gunma 371, Japan Received May 2, 1991/Revised, accepted June 17, 1991
Summary. Granulovacuolar degeneration ( G V D ) in the hippocampal pyramidal neurons of Alzheimer-type dementia was examined. Immunohistochemical examinations showed that the majority of centrally located granules were positive for ubiquitin. Based on electron microscopic observations, morphogenesis of G V D is considered to be as follows. Slight-to-moderate amounts of electron-dense material appear in the cytoplasm at the early stage, and are then surrounded and demarcated by a two-layered m e m b r a n e (probably from smooth endoplasmic reticulum). Following this some inner material is digested forming floccular and liquidlike materials, while undigested material remains as coarse electron-dense granules. Specifically, granulovacuoles are considered to be an age-related special type of autophagosome. Analytical electron microscopy disclosed that the granules in G V D contained some aluminum.
or the content of the vacuoles. Some investigators suggested that G V D represented autophagic abnormalities [17], or that granulovacuoles bore some resemblance to the lipofuscin deposits [8]. Recent immunohistochemical studies have shown that the granules of G V D react with antibodies to tubulin [15], neurofilamerits [5, 9] and ubiquitin [11], but negative data have also been reported [12]. Recently, we were able to observe G V D in well-fixed samples of hippocampus from ATD, and found a variety of granulovacuolar morphologies, which suggest the process of G V D formation. We also examined granulovacuoles by immunohistochemical stainings with several antibodies and by analytical electron microscopy.
Key words: Ubiquitin - granulovacuolar degeneration -
Brains taken at autopsy of three patients with clinical and pathological features of ATD were examined by light and electron microscopy. After formalin fixation, samples from hippocampal areas were embedded in paraffin. H&E, Kltiver-Barrera, PAS, phosphotungtic acid-hematoxylin (PTAH), Mallory, Congo red, Bodian and modified Bielschowsky methods were carried out on 5-~m sections. For immunohistochemistry, 5 ~tm deparaffinized sections were incubated with the diluted primary antibodies overnight at room temperature, then stained by the avidin-biotin-peroxidas (ABC) method (Vector Lab.). The antibodies employed in the present study were: a monoclonal anti-ubiquitin (Chemicon, 1:2000), polyclonal anti-ubiquitin (Dakopatts, 1:200 and Sigma, 1:20), anti-c~ tubulin (Bio-Yeda, 1:200), anti-j3 tubulin (Bio-Makor, 1:200), anti-neurofilament (160K, 200K, Dr. H. Yamaguchi), monoclonal anti-neurofilament (phosphorylated, Dr. Y. Nakazato), anti-tau (Dr.Y. Ihara), anti paired hilical filament (Pt-IF; Dr.Y. Ihara), anti-glial fibrillary acidic protein (GFAE Dr. H. Yamaguchi), anti-desmin (Dakopatts), anti-actin (Bio-Makor), and antisynthetic ~ protein (1-28, pretreatment with formic acid) antibodies. For electron microscopic examination, hippocampal areas were fixed in 4 % paraformaldehyde mixed with 1% glutaraldehyde and refixed in 1% osmic acid, then embedded in Epon. Analytical electron microscopy was also carried out on unstained ultrathin sections (JEOL, Tokyo).
ageing
T h e three main pathological hallmarks of Alzheimertype dementia (ATD) are Alzheimer neurofibrillary tangles (NFT), senile plaques and granulovacuolar degeneration ( G V D ) . G V D was first described by Simchowicz in 1911 [16]. The G V D manifests as small inclusions, 3- to 5-~m diameter spherical vacuoles containing centrally located argentophilic and hematoxyphilic granules,which are found most commonly in the hippocampal pyramidal neurons [2, 16, 19, 20]. On electron microscopy, granulovacuoles appear as cytoplasmic m e m b r a n e - b o u n d e d inclusions containing a dense core of granular material [8, 14, 17]. However, previous light and electron microscopic examinations gave few clues as to the origin of the structural changes Offprint requests to: K. Okamoto (adress see above)
Materials and methods
341
Results
Light microscopy Many granulovacuoles were seen in hippocampal pyramidal neurons. Granules of GVD were argentophilic and hematoxyphilic, but negative for PAS and Congo red. Immunohistochemical examinations disclosed that the majority of the granules of GVD in our three patients showed positive reactions for all three antiubiquitin antisera (Fig. 1). Tubulin-like immunoreactivity was detected in some granules by (~ and ~ tubulin immunostainings, but it was weak and similar to that shown in the neuronal cytoplasm and processes. Other antibodies, including neurofilament, tau, PHF and actin, failed to stain the granules of GVD. Some neurons with granulovacuoles also contained NFT which were stained with ubiquitin, tau and PHF.
Fig. 1. Granules of granulovacuolar degeneration (GVD) in the
hippocampal neurons are stained with ubiquitin (arrows). Alzheimer neurofibrillary tangles are also stained (arrowhead).Ubiquitin (monoclonal) immunostaining. • 400
Electron microscopy
Discussion
Many granulovacuoles were seen in the hippocampal neurons (Figs. 2-9). As has been previously reported [8], these consist of membrane-bounded inclusions containing an irregular cluster of electron-dense granular material. However, we also observed many other morphologies suggesting the process of granulovacuolar formation (Fig. 2). Some vacuoles contained slightto-moderate amounts of electron-dense material including a few cellular organelles partially surrounded by a two-layered membrane (not a single membrane), but with direct connections with the neuronal cytoplasm (Figs. 3,4). The distance between the two membranes was variable, and the ribosomes were not attached to the membranes. Atzheimer NFT located close to some granulovacuoles (Fig. 5), but neither NFT nor filamentous structures were seen inside granulovacuoles. A few granulovacuoles were located adjacent to lipofuscin granules and were surrounded by the same membrane (Fig. 6), but the majority of granulovacuoles were not in association with lipofuscin granules. Occasionally, Golgi apparatus (Fig. 7) and an accumulation of small vesicles (Fig. 8) were located close to a granulovacuole. A granulovacuole, in which the inner material was floccular of liquid-like, the electron-dense material coarse and dense, and in which the inner membrane was undulated or had disappeared in some places is shown in Fig. 9.
When GVD is observed, it is almost always located in pyramidal neurons of Sommer's sector and adjacent areas, and a single neuron may contain a few or many granulovacuoles. The number of granulovacuoles increases with age, and much more so in patients with ATD [2, 19, 20]. Some researchers, on the basis of morphology, have suggested that granulovacuoles are lysosomal in nature [17], but the exact histological origin of GVD remains unknown. While electron micrographs of autopsy tissues have shown that a granulovacuole consists of a vacuole containing clear material and a core of dense granular material bounded by a "unit membrane" [8, 17], poor fixation has usually precluded satisfactory analysis of morphogenesis. Oyanagi and Ikuta [14] examined the fine structures of granulovacuoles that appeared as large vacuoles having undulated membranes close to and inside their limiting membranes. In addition, they observed that a small part of the neuronal cytoplasm, containing electron-dense material and lysosomes, was occasinally partially or almost completely surrounded by flattened cytoplasmic vacuoles. The authors suggested that this morphology may represent an early event in the granulovacuolar formation and may precede digestion of the demarcated part of the cytoplasm. They further suggested that this process is comparable to that of autophagocytosis in cells in general. The term autophagic vacuoles is used for secondary lysosomes containing morphologically recognizable cytoplasmic components [1, 4]. Two types of autophagic vacuoles are recognized: (1) the autophagosome, which is defined as a double-membrane-limited body; and (2) the autolysosome, which is a single- or double-membrane-limited body containing hydrolase activity [1]. The origin of the membrane enclosing autophagic vacuoles has been studied and several possibilities have been suggested [1]. It was considered from morphological and histochemical
Analytical electron microscopy Analytical electron microscopy showed a small peak on the aluminum area for the electron-dense granules of GVD (Fig. 10). Although several other chemical elements were detected by this method, detection was mainly due to the copper grid, fixative agents and embedding materials used.
342
Figs. 2-9. A variety of morphologies of G V D
Figs. 3, 4. Slight-to-moderate amounts of electron-dense material
Fig. 2. Many granulovacuoles seen u n d e r low magnification. Morphology of granules is somewhat different in each vacuole. N; nucleus • 12,600
are surrounded by a two-layered m e m b r a n e which closely resembles smooth endoplasmic reticulum. Direct connections between the inner material and neuronal cytoplasm are dearly seen. Filamentous structures are not seen in the inner material. 3 x 24,400; 4 • 17,200
343
Fig. 5. The granules contain some vesicles. Alzheimer neurofibrillary tangles are seen outside this granulovacuole, x 53,400 Fig. 6. Small lipofuscin granule and granulovacuole are surrounded by the same membrane (arrow). • 34,900
approaches that the double-membrane-limited sac forming the wall of the early autophagic vacuole in the liver originated from smooth endoplasmic reticulum [1]. Terry et al. [18] studied autophagic vacuoles in neurons treated with spindle inhibitors, and they observed that some of the compacted membranes of the rough endoplasmic reticulum formed closed circles, becoming autophagic vacuoles. As the membranes compacted, the
Fig. 7. Golgi apparatus is close to this granulovacuole. • 20,100 Fig. 8. Several small vesicles (arrow) are also seen adjacent to granulovacuole, x 18,500
ribosomes became detached. In the early stages, the enclosed material appeared identical to the surrounding cytoplasm, and later stages showed degradation of the vacuolar contents [18]. Brosnan et al. [3] examined chlorpromazine-induced granules in cultured neurons, and they observed that autophagic vacuoles often exhibit a bounding double m e m b r a n e suggestive of formation from a cisterna of endoplasmic reticulum.
344
Fig. 9. Electron-dense material is coarse, and the inner membranes undulate or have disappeared in some parts, x 22,500
Fig. 10. Analytical electron microscopy of a granule of GVD. Letters at each peak are abbreviations of chemical elements detected by this method: C, carbon; O, oxygen; Al, aluminum; Cu, copper; Os, osmium; P, potassium; S, sulfur; Cl, chlorine. Detection of chemical elements other than aluminum was mainly due to the copper grid, fixative agents and embedding materials used. In our studies, we were able to observe well-fixed samples and to observe morphological variation which suggested the process of granulovacuolar formation. On the basis of our observations, we suggest the following mechanism for the process of granulovacuolar forma-
tion. On appearance of slight-to-moderate quantities of electron-dense material without filamentous structures in the cytoplasm, a membranous system surrounds and demarcates the material, which may be noxious to the neuron. The membrane is composed of a two-layered unit described by Oyanagi and Ikuta [14], the morphology of which strongly suggests that it originates from smooth endoplasmic reticulum. Some of the demarcated material is gradually digested becoming floccular or liquid-like, while the undigested material remains as coarse electron-dense granules. The inner membrane of the two-layered unit becomes undulated or is digested and disappears at the advanced stage. The morphology of GVD is similar to autophagic vacuoles in general [1, 3, 4, 17], therefore granulovacuoles are thought to be an age-related special type of autophagic vacuole, and the granules may be residual bodies. While autophagic vacuoles usually show evidence of enzyme activity and, therefore, qualify as secondary lysosomes [4], there are few histochemical studies of granulovacuoles. Hydrolytic enzymes including acid phosphatase were present in granulovacuoles [10]. The histochemical characteristics of granulovacuoles require further study. On the other hand, several immunohistochemical studies of granulovaculoes have been reported recently. Price et al. [15] reported that tubulin-like immunoreactivity was present in granules in vacuoles, whereas other cytoskeletal antigens, including microtubule-associated proteins (MAP 2) and neurofilaments, were not demon-
345 strated in granules. O n e of the antibodies to neurofilaments was r e p o r t e d to react with the granular components of granulovacuoles [9]. Dickson et al. [5] reported that a monoclonal antibody which recognizes a phosphorylated epitope in NFT, neurofilaments and tau proteins i m m u n o s t a i n e d G V D , and suggested that granulovacuoles contain p h o s p h o r y l a t e d protein due to autophagy of p h o s p h o r y l a t e d perikaryal protein, which appears to be increased in ATD. L o v e et al. [11] observed that the granules in vacuoles were stained with anti-ubiquitin, but M a n e t t o et al. [12] described that ubiquitin was not detected in granulovacuoles. In our study, tubulin-like immunoreactivity was seen in some granules in vacuoles, but the degree was weak and similar to that in neuronal cytoplasm and dendrites. This result can be u n d e r s t o o d if the initial stage of granulovacuolar f o r m a t i o n is considered to be the demarcation of an area of the cytoplasm. I m m u n o r e a c tivity to neurofilaments, tau, Pt-IF, actin and [3 protein was not d e m o n s t r a t e d in the granules. The Granules in vacuoles were stained with antiubiquitin antibodies. Ubiquitin, a protein implicated in non-lysosomal degradation of a b n o r m a l and short-lived cellular proteins, has recently been associated with a b n o r m a l filaments characteristic of neurodegenerative disease and age-related axonal dystrophy [6, 12]. Ubiquitin-immunoreactive structures in dystrophic neurites resemble lysosomal residual bodies, and it is reported that ubiquitinated proteins m a y also be accessible to lysosomal degradative pathways [6]. Analytical electron microscopy d e m o n s t r a t e d that the granules of G V D contained some degree of aluminum. Recently, G o o d and Perl [7] d e m o n s t r a t e d that the G V D granules in b o t h Alzheimer's disease and G u a m amyotrophic lateral sclerosis/parkinsonism d e m e n t i a complex showed extremely p r o m i n e n t aluminumrelated signals by laser microprobe mass analysis. M c D e r m o t t et al. [13] r e p o r t e d that the brain aluminum concentration, as assayed by atomic absorption spectroscopy, increases with age and that the h i p p o c a m p u s has the highest concentratoin of aluminum in b o t h A T D patients and a g e - m a t c h e d controls. In conclusion, we consider that granulovacuoles are an age-related special type of a u t o p h a g o s o m e , but we cannot explain why they are generally confined to h i p p o c a m p a l pyramidal neurons. T h e nature of the material which m a y have b e e n the first to a p p e a r in the cytoplasm at the early stage of G V D f o r m a t i o n requires further study.
Acknowledgements. The authors thank Dr.Yasuo Ihara, Dr.Yoichi Nakazato and Dr. Haruyasu Yamaguchi for their kind provision of antibodies.
References 1. Arstila AU, Trump BF (1968) Studies on cellular autophagocytosis. The formation of autophagic vacuoles in the liver after gIucagon administration. Am J Pathol 53:687-733
2. Bail MJ, Lo P (1977) Granulovacuolar degeneration in aging brain and in dementia. J Neuropathol Exp Neurol 36: 474 487 3. Brosnan CF, Bunge MB, Murray MR (1970) The response of lysosomes in cultured neurons to chlorporomazine. J Neuropathol Exp Neurol 29:337-353 4. De Duve C (1967) Lysosomes and phagosomes. Protoplasma 63:95-98 5. Dickson DW, Ksiezak-Reding H, Davis R Yen S-H (1987) A monoclonal antibody that recognizes a phosphorylated epitope in Alzheimer neurofibriallary tangles, neurofilaments and tau proteins immunostains granulovacuolar degeneration. Acta Neuropathol (Berl) 73:254-258 6. Dickson DW,Wertkin A, KressY, Ksiezak-Reding H,Yen S-H (1990) Ubiquitin immunoreactive in normal human brains. Distribution and developmental aspects. Lab Invest 63: 87-99 7. Good PF, Perl DP (1990) Laser microprobe mass analysis (LAMMA) evidence of aluminum accumulation in the granules of granulovacuolar degeneration of Alzheimer's disease (AD) and Guam ALS/parkinsonism dementia complex (ALSPDC) (abstract). J Neuropathol Exp Neurol 49:317 8. Hirano A, Dembitzer HM, Kurland LT, Zimmerman HM (1968) The fine structure of some intraganglionic alteration. Neurofibrillary tangles, granulovacuolar bodies and "rod-like" structures as seen in Guam amyotropbic lateral sclerosis and Parkinson-dementia complex. J Neuropathol Exp Neurol 27: 167-182 9. Kahn J, Anderton BH, Probst A, Ulrich J, Esiri M (1985) Immunohistological study of granulovacuolar degeneration using monoclonal antibodies to neurofilaments. J Neurol Neurosurg Psychiatry 48:924-926 10. Krigman MR, Feldman RG, Bensch K (1965) Alzheimer's presenile dementia. A histochemical and electron microscopic study. Lab Invest 14:381-396 11. Love S, SaitohT, Quijada S, Cole GM,Terry RD (1988) Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. J Neuropathol Exp Neurol 47: 393-405 12. Manetto V, Perry G,Tabaton M, Mulvihill R Fried VA, Smith HT, Gambetti P, Autilio-Gambetti L (1988) Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases. Proc Natl Acad Sci USA 85: 4501-4505 13. McDermott JR, Smith AI, Iqbal K, Wisniewski HM (1979) Brain aluminum in aging and Alzheimer disease. Neurology 29:809-814 14. Oyanagi S, Ikuta F (1974) An ultrastructural observation on granulovacuolar degenerations in reference to the process of their formation. Brain Nerve (Tokyo) 26:783-788 15. Price DL, Altschuler RJ, Struble RG, Casanova MF, Cork LC, Murphy DB (1986) Sequestration of tubulin in neurons in Alzheimer's disease. Brain Res 385:305-310 16. Simchowicz T (1911) Histologische Studien fiber die senile Demenz. Histogische und Histopathologische Arbeiten tiber die Grol3hirnrinde 4:267-444 17. Terry RD, Wisniewski HM (1972) Ultrastructure of senile dementia and of experimental analogs. In: Gaintz CM (ed) Aging and the brain. Plneum Press, New York pp 89-115 18. Terry RD, Wisniewski H, Johnson AB (1970) Studies on the formation of autophagic vacuoles in neurons treated with spindle inhibitors (colchicine and vinblastine) (abstract). J Neuropathol Exp Neurol 29:142-143 19. Tomlinson BE, Kitchener D (1972) Granulovacuolar degeneration of hippocampal pyramidal cells. J Pathol 106: 165-185 20. Woodard JS (1962) Clinicopathologic significance of granulovacuolar degeneration in Alzheimer's disease. J Neuropathol Exp Neurol 21:85-91
Acta Heuropatholog,ca O Springer-Verlag 1991
Reexamination of granulovacuolar degeneration K. Okamoto, S. Hirai, T. Iizuka, T. Yanagisawa, and M. Watanabe Department of Neurology, Gunma University School of Medicine, Maebashi, Gunma 371, Japan Received May 2, 1991/Revised, accepted June 17, 1991
Summary. Granulovacuolar degeneration ( G V D ) in the hippocampal pyramidal neurons of Alzheimer-type dementia was examined. Immunohistochemical examinations showed that the majority of centrally located granules were positive for ubiquitin. Based on electron microscopic observations, morphogenesis of G V D is considered to be as follows. Slight-to-moderate amounts of electron-dense material appear in the cytoplasm at the early stage, and are then surrounded and demarcated by a two-layered m e m b r a n e (probably from smooth endoplasmic reticulum). Following this some inner material is digested forming floccular and liquidlike materials, while undigested material remains as coarse electron-dense granules. Specifically, granulovacuoles are considered to be an age-related special type of autophagosome. Analytical electron microscopy disclosed that the granules in G V D contained some aluminum.
or the content of the vacuoles. Some investigators suggested that G V D represented autophagic abnormalities [17], or that granulovacuoles bore some resemblance to the lipofuscin deposits [8]. Recent immunohistochemical studies have shown that the granules of G V D react with antibodies to tubulin [15], neurofilamerits [5, 9] and ubiquitin [11], but negative data have also been reported [12]. Recently, we were able to observe G V D in well-fixed samples of hippocampus from ATD, and found a variety of granulovacuolar morphologies, which suggest the process of G V D formation. We also examined granulovacuoles by immunohistochemical stainings with several antibodies and by analytical electron microscopy.
Key words: Ubiquitin - granulovacuolar degeneration -
Brains taken at autopsy of three patients with clinical and pathological features of ATD were examined by light and electron microscopy. After formalin fixation, samples from hippocampal areas were embedded in paraffin. H&E, Kltiver-Barrera, PAS, phosphotungtic acid-hematoxylin (PTAH), Mallory, Congo red, Bodian and modified Bielschowsky methods were carried out on 5-~m sections. For immunohistochemistry, 5 ~tm deparaffinized sections were incubated with the diluted primary antibodies overnight at room temperature, then stained by the avidin-biotin-peroxidas (ABC) method (Vector Lab.). The antibodies employed in the present study were: a monoclonal anti-ubiquitin (Chemicon, 1:2000), polyclonal anti-ubiquitin (Dakopatts, 1:200 and Sigma, 1:20), anti-c~ tubulin (Bio-Yeda, 1:200), anti-j3 tubulin (Bio-Makor, 1:200), anti-neurofilament (160K, 200K, Dr. H. Yamaguchi), monoclonal anti-neurofilament (phosphorylated, Dr. Y. Nakazato), anti-tau (Dr.Y. Ihara), anti paired hilical filament (Pt-IF; Dr.Y. Ihara), anti-glial fibrillary acidic protein (GFAE Dr. H. Yamaguchi), anti-desmin (Dakopatts), anti-actin (Bio-Makor), and antisynthetic ~ protein (1-28, pretreatment with formic acid) antibodies. For electron microscopic examination, hippocampal areas were fixed in 4 % paraformaldehyde mixed with 1% glutaraldehyde and refixed in 1% osmic acid, then embedded in Epon. Analytical electron microscopy was also carried out on unstained ultrathin sections (JEOL, Tokyo).
ageing
T h e three main pathological hallmarks of Alzheimertype dementia (ATD) are Alzheimer neurofibrillary tangles (NFT), senile plaques and granulovacuolar degeneration ( G V D ) . G V D was first described by Simchowicz in 1911 [16]. The G V D manifests as small inclusions, 3- to 5-~m diameter spherical vacuoles containing centrally located argentophilic and hematoxyphilic granules,which are found most commonly in the hippocampal pyramidal neurons [2, 16, 19, 20]. On electron microscopy, granulovacuoles appear as cytoplasmic m e m b r a n e - b o u n d e d inclusions containing a dense core of granular material [8, 14, 17]. However, previous light and electron microscopic examinations gave few clues as to the origin of the structural changes Offprint requests to: K. Okamoto (adress see above)
Materials and methods
341
Results
Light microscopy Many granulovacuoles were seen in hippocampal pyramidal neurons. Granules of GVD were argentophilic and hematoxyphilic, but negative for PAS and Congo red. Immunohistochemical examinations disclosed that the majority of the granules of GVD in our three patients showed positive reactions for all three antiubiquitin antisera (Fig. 1). Tubulin-like immunoreactivity was detected in some granules by (~ and ~ tubulin immunostainings, but it was weak and similar to that shown in the neuronal cytoplasm and processes. Other antibodies, including neurofilament, tau, PHF and actin, failed to stain the granules of GVD. Some neurons with granulovacuoles also contained NFT which were stained with ubiquitin, tau and PHF.
Fig. 1. Granules of granulovacuolar degeneration (GVD) in the
hippocampal neurons are stained with ubiquitin (arrows). Alzheimer neurofibrillary tangles are also stained (arrowhead).Ubiquitin (monoclonal) immunostaining. • 400
Electron microscopy
Discussion
Many granulovacuoles were seen in the hippocampal neurons (Figs. 2-9). As has been previously reported [8], these consist of membrane-bounded inclusions containing an irregular cluster of electron-dense granular material. However, we also observed many other morphologies suggesting the process of granulovacuolar formation (Fig. 2). Some vacuoles contained slightto-moderate amounts of electron-dense material including a few cellular organelles partially surrounded by a two-layered membrane (not a single membrane), but with direct connections with the neuronal cytoplasm (Figs. 3,4). The distance between the two membranes was variable, and the ribosomes were not attached to the membranes. Atzheimer NFT located close to some granulovacuoles (Fig. 5), but neither NFT nor filamentous structures were seen inside granulovacuoles. A few granulovacuoles were located adjacent to lipofuscin granules and were surrounded by the same membrane (Fig. 6), but the majority of granulovacuoles were not in association with lipofuscin granules. Occasionally, Golgi apparatus (Fig. 7) and an accumulation of small vesicles (Fig. 8) were located close to a granulovacuole. A granulovacuole, in which the inner material was floccular of liquid-like, the electron-dense material coarse and dense, and in which the inner membrane was undulated or had disappeared in some places is shown in Fig. 9.
When GVD is observed, it is almost always located in pyramidal neurons of Sommer's sector and adjacent areas, and a single neuron may contain a few or many granulovacuoles. The number of granulovacuoles increases with age, and much more so in patients with ATD [2, 19, 20]. Some researchers, on the basis of morphology, have suggested that granulovacuoles are lysosomal in nature [17], but the exact histological origin of GVD remains unknown. While electron micrographs of autopsy tissues have shown that a granulovacuole consists of a vacuole containing clear material and a core of dense granular material bounded by a "unit membrane" [8, 17], poor fixation has usually precluded satisfactory analysis of morphogenesis. Oyanagi and Ikuta [14] examined the fine structures of granulovacuoles that appeared as large vacuoles having undulated membranes close to and inside their limiting membranes. In addition, they observed that a small part of the neuronal cytoplasm, containing electron-dense material and lysosomes, was occasinally partially or almost completely surrounded by flattened cytoplasmic vacuoles. The authors suggested that this morphology may represent an early event in the granulovacuolar formation and may precede digestion of the demarcated part of the cytoplasm. They further suggested that this process is comparable to that of autophagocytosis in cells in general. The term autophagic vacuoles is used for secondary lysosomes containing morphologically recognizable cytoplasmic components [1, 4]. Two types of autophagic vacuoles are recognized: (1) the autophagosome, which is defined as a double-membrane-limited body; and (2) the autolysosome, which is a single- or double-membrane-limited body containing hydrolase activity [1]. The origin of the membrane enclosing autophagic vacuoles has been studied and several possibilities have been suggested [1]. It was considered from morphological and histochemical
Analytical electron microscopy Analytical electron microscopy showed a small peak on the aluminum area for the electron-dense granules of GVD (Fig. 10). Although several other chemical elements were detected by this method, detection was mainly due to the copper grid, fixative agents and embedding materials used.
342
Figs. 2-9. A variety of morphologies of G V D
Figs. 3, 4. Slight-to-moderate amounts of electron-dense material
Fig. 2. Many granulovacuoles seen u n d e r low magnification. Morphology of granules is somewhat different in each vacuole. N; nucleus • 12,600
are surrounded by a two-layered m e m b r a n e which closely resembles smooth endoplasmic reticulum. Direct connections between the inner material and neuronal cytoplasm are dearly seen. Filamentous structures are not seen in the inner material. 3 x 24,400; 4 • 17,200
343
Fig. 5. The granules contain some vesicles. Alzheimer neurofibrillary tangles are seen outside this granulovacuole, x 53,400 Fig. 6. Small lipofuscin granule and granulovacuole are surrounded by the same membrane (arrow). • 34,900
approaches that the double-membrane-limited sac forming the wall of the early autophagic vacuole in the liver originated from smooth endoplasmic reticulum [1]. Terry et al. [18] studied autophagic vacuoles in neurons treated with spindle inhibitors, and they observed that some of the compacted membranes of the rough endoplasmic reticulum formed closed circles, becoming autophagic vacuoles. As the membranes compacted, the
Fig. 7. Golgi apparatus is close to this granulovacuole. • 20,100 Fig. 8. Several small vesicles (arrow) are also seen adjacent to granulovacuole, x 18,500
ribosomes became detached. In the early stages, the enclosed material appeared identical to the surrounding cytoplasm, and later stages showed degradation of the vacuolar contents [18]. Brosnan et al. [3] examined chlorpromazine-induced granules in cultured neurons, and they observed that autophagic vacuoles often exhibit a bounding double m e m b r a n e suggestive of formation from a cisterna of endoplasmic reticulum.
344
Fig. 9. Electron-dense material is coarse, and the inner membranes undulate or have disappeared in some parts, x 22,500
Fig. 10. Analytical electron microscopy of a granule of GVD. Letters at each peak are abbreviations of chemical elements detected by this method: C, carbon; O, oxygen; Al, aluminum; Cu, copper; Os, osmium; P, potassium; S, sulfur; Cl, chlorine. Detection of chemical elements other than aluminum was mainly due to the copper grid, fixative agents and embedding materials used. In our studies, we were able to observe well-fixed samples and to observe morphological variation which suggested the process of granulovacuolar formation. On the basis of our observations, we suggest the following mechanism for the process of granulovacuolar forma-
tion. On appearance of slight-to-moderate quantities of electron-dense material without filamentous structures in the cytoplasm, a membranous system surrounds and demarcates the material, which may be noxious to the neuron. The membrane is composed of a two-layered unit described by Oyanagi and Ikuta [14], the morphology of which strongly suggests that it originates from smooth endoplasmic reticulum. Some of the demarcated material is gradually digested becoming floccular or liquid-like, while the undigested material remains as coarse electron-dense granules. The inner membrane of the two-layered unit becomes undulated or is digested and disappears at the advanced stage. The morphology of GVD is similar to autophagic vacuoles in general [1, 3, 4, 17], therefore granulovacuoles are thought to be an age-related special type of autophagic vacuole, and the granules may be residual bodies. While autophagic vacuoles usually show evidence of enzyme activity and, therefore, qualify as secondary lysosomes [4], there are few histochemical studies of granulovacuoles. Hydrolytic enzymes including acid phosphatase were present in granulovacuoles [10]. The histochemical characteristics of granulovacuoles require further study. On the other hand, several immunohistochemical studies of granulovaculoes have been reported recently. Price et al. [15] reported that tubulin-like immunoreactivity was present in granules in vacuoles, whereas other cytoskeletal antigens, including microtubule-associated proteins (MAP 2) and neurofilaments, were not demon-
345 strated in granules. O n e of the antibodies to neurofilaments was r e p o r t e d to react with the granular components of granulovacuoles [9]. Dickson et al. [5] reported that a monoclonal antibody which recognizes a phosphorylated epitope in NFT, neurofilaments and tau proteins i m m u n o s t a i n e d G V D , and suggested that granulovacuoles contain p h o s p h o r y l a t e d protein due to autophagy of p h o s p h o r y l a t e d perikaryal protein, which appears to be increased in ATD. L o v e et al. [11] observed that the granules in vacuoles were stained with anti-ubiquitin, but M a n e t t o et al. [12] described that ubiquitin was not detected in granulovacuoles. In our study, tubulin-like immunoreactivity was seen in some granules in vacuoles, but the degree was weak and similar to that in neuronal cytoplasm and dendrites. This result can be u n d e r s t o o d if the initial stage of granulovacuolar f o r m a t i o n is considered to be the demarcation of an area of the cytoplasm. I m m u n o r e a c tivity to neurofilaments, tau, Pt-IF, actin and [3 protein was not d e m o n s t r a t e d in the granules. The Granules in vacuoles were stained with antiubiquitin antibodies. Ubiquitin, a protein implicated in non-lysosomal degradation of a b n o r m a l and short-lived cellular proteins, has recently been associated with a b n o r m a l filaments characteristic of neurodegenerative disease and age-related axonal dystrophy [6, 12]. Ubiquitin-immunoreactive structures in dystrophic neurites resemble lysosomal residual bodies, and it is reported that ubiquitinated proteins m a y also be accessible to lysosomal degradative pathways [6]. Analytical electron microscopy d e m o n s t r a t e d that the granules of G V D contained some degree of aluminum. Recently, G o o d and Perl [7] d e m o n s t r a t e d that the G V D granules in b o t h Alzheimer's disease and G u a m amyotrophic lateral sclerosis/parkinsonism d e m e n t i a complex showed extremely p r o m i n e n t aluminumrelated signals by laser microprobe mass analysis. M c D e r m o t t et al. [13] r e p o r t e d that the brain aluminum concentration, as assayed by atomic absorption spectroscopy, increases with age and that the h i p p o c a m p u s has the highest concentratoin of aluminum in b o t h A T D patients and a g e - m a t c h e d controls. In conclusion, we consider that granulovacuoles are an age-related special type of a u t o p h a g o s o m e , but we cannot explain why they are generally confined to h i p p o c a m p a l pyramidal neurons. T h e nature of the material which m a y have b e e n the first to a p p e a r in the cytoplasm at the early stage of G V D f o r m a t i o n requires further study.
Acknowledgements. The authors thank Dr.Yasuo Ihara, Dr.Yoichi Nakazato and Dr. Haruyasu Yamaguchi for their kind provision of antibodies.
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