Immunocytochemical and Ultrastructural Studies of Pick's Disease Shigeo Murayama, MD,"? Hiroshi Mori, PhDJ Yasuo Ihara, MD,S and Masanori Tomonaga, MD"

Cerebral cortical changes in 10 cases with Picks disease were studied immunocytochemically and ultrastructurally. All cases contained Picks argentophilic bodies and ballooned neurons. The antibodies against phosphorylated tau proteins that intensely stained all Pick bodies recognized numerous neuronal processes around Pick body-bearing cells and focal portions in the perikarya of ballooned neurons. Monoclonal and polyclonal anti-ubiquitin antibodies stained not only some Pick bodies with variable intensity, but also the perikarya of all ballooned neurons. Ultrastructurally, Pick bodies consisted of accumulation of randomly oriented, approximately 15-nm straight filaments and paired twisted profiles with a minimal diameter of 13 nm, maximal diameter of 26 nm, and twist periodicity of 120 nm. These Pick body-type filaments were also observed in the perikarya of ballooned neurons and neuronal processes around Pick body-bearing cells. Our studies demonstrate, for the first time, the characteristic pathological feature of neuropil in Picks disease. Pick body-bearing cells and ballooned neurons show unique immunocytochemical and ultrastructural properties that may be a clue to the pathogenesis of Picks disease. Murayama S, Mori H, Ihara Y, Tomonaga M. Immunocytochemical and ultrastructural studies of Pick's disease. Ann Neurol 1990;27:394-405

The pathological criteria of Pick's disease (PD) are (1) circumscribed cortical atrophy [l}, and (2) the absence of Alzheimer's neurofibrillary tangles o r senile plaques in the affected cortex [2, 31. P D shows two characteristic neuronal changes: Pick's argentophilic bodies (PBs) in small-sized neurons 147 and ballooned neurons (BNs) in large neurons in the third and fifth layer of the affected cortex [S-77. The relationship between PBs and BNs has been one of the unsolved problems in PD. PB-bearing cells and BNs appear similar in Nissl-stained sections [S] but different in Bodian-stained sections 131. Schneider postulated that BNs were a precursor of PB-bearing cells 191, while others denied the evolution of PB from BNs on the basis of routine light-microscopical observations {lo}. There is also a confusion about the definition of PD. Some patients who lacked characteristic neuronal changes (PB and BN) have been given a diagnosis of PD on the basis of gross pathological findings [lo]. It is probable that heterogeneous disorders have been categorized as P D Ell, 121. In this paper, we restrict PD to mean the cases that have both PBs and BNs, in order to avoid further confusion. The purpose of this study is to characterize PBbearing cells and BNs immunocytochemically and ul-

trastructurally. We also study the cases with circumscribed cortical atrophy without PB or B N (questionable PD).

From the "Department of Neuropathology, Institute of Brain Research, Faculty of Medicine, University of Tokyo, Tokyo; the tDivision of Neuropathology, Department of Pathology, University of North Carolina at Chapel Hill, Chapel Hill, NC; and the *Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan.

Received May 12, 1989, and in revised form Aug 2. Accepted for publication Oct 6. 1989.

394

Materials and Methods Tissue Sources Brains from 10 patients with PD were studied. The age, sex, and clinical course of each are listed in Table 1 (Cases 1-10), One (Case 6) was of European ancestry and the others were of Japanese ancestry. The brains were fixed in formalin and several regions were embedded in paraffin. For routine neuropathological study, the sections were stained with hematoxylin-eosin, Nissl, myelin, silver, and Holzer stains. Four brains from patients with questionable PD were also examined. Their age, sex, and clinical course are also listed in Table 1 (Cases 11-14). Various areas of the brain from 5 patients with Alzheimer's disease (AD) and 5 with progressive supranuclear palsy were studied for comparison. BNs were also examined in 2 cases with pellagra encephalomyelopathy, 2 cases with Creutzfeldt-Jacob disease, and 2 cases with subcortical infarction. Various regions of the brains from 10 neurologically and neuropathologically normal subjects were also studied.

Primaly Antibodies The antibodies used in the present study were as follows: affiniry-purified antibodies to phosphorylated tau (anti-ptau)

Address correspondence to Dr Murayma, Division of NeuropaD~~~~~~~ of Pathology, CB #7525, BrinkhouS-Bullitt &,logy, Building, #409, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7525.

Copyright 0 1970 by the American Neurological Association

(Aldrich Chemical, Milwaukee, WI) for 30 minutes, and reacted with diluted antibodies for 48 hours, using the floating method. All sections were subsequently reacted with diluted peroxidase-conjugated secondary antibody (1 :200) (Tago, Burlingame, CA) and visualized with diaminobenzidine. After microscopic observation, the sections were embedded in epoxy resin and ultrathin sections were obtained.

Results Neuropathology All 10 brains showed circumscribed cortical atrophy. The brain weights and topography of the cortical atrophy are summarized in Table 1. In the atrophic regions, severe neuronal loss, degeneration of subconical white matter, severe cortical and subcortical gliosis, and the existence of PBs and BNs were characteristic features. Case 6 was associated with abundant Alzheimer's neurofibrillary tangIes and senile plaques in the hippocampus and subiculum, and Case 9 contained progressive supranuclear palsy-type neurofibrillary tangles in brainstem nuclei, in addition to typical changes of PD (K. Arima, personal communication, 1988). PBs were observed as round argentophilic inclusions with Bodian stain (Fig la). Some PBs had a kidneyshaped or crescent-shaped form. In toluidine bluestained sections, PBs were demarcated, round, and lightly stained (Fig 3a). PBs were abundant in the hippocampus in all cases except Case 8, in which PBs were only present in small neurons of the cingulate gyrus. Many Hirano bodies and granulovacuolar degenerations were observed with PBs in hippocampus. BNs were easily identified with Nissl stain as swollen chromatolytic neurons. BNs often contained intracytoplasmic vacuoles (Fig 2a) and granulovacuolar degenerations (see Fig 2e). BNs were observed in the affected neocortex, cingulate gyrus, and subiculum. Argentophilia within the swollen perikarya was generally limited to small focal areas (see Fig 2a). In toluidine blue-stained sections, BNs were characterized by pale ballooned cytoplasm and eccentric nuclei (Fig 4a). The 4 brains with questionable PD showed neuronal loss and gliosis in atrophic areas. Neither PBs nor BNs were observed. lmmunocytocbemistty

All the PBs were intensely stained with anti-ptau (see Fig Id) and antiPHF (not illustrated). PBs showed a homogeneous or fibrillary pattern. In addition, numerous cell processes were visualized around PB-bearing cells (see Fig Id). A small focal region was stained with the antibodies in the perikarya of normal-appearing neurons in the vicinity of the PB-bearing cells (not illustrated). The cytoplasm of many BNs also focally stained

ANTI-PTAU A N D ANTI-PHF.

396 Annals of Neurology Vol 2 7 No 4 April 1990

with the antibodies (see Figs 2b and 2c). These focal areas corresponded to the argentophilic region on adjacent sections with Bodian stain (see Fig 2a). Of the 4 cases of questionable PD, neuronal structures staining with anti-ptau or anti-PHF were limited to a few neurohbrillary tangles of the AD type in the hippocampus and subiculum in Case 14. Neurofibrillary tangles of the progressive supranuclear palsy type were also recognized by the antibodies. The antibodies did not stain the perikarya of BNs in disorders other than PD. ANTI-UBIQUITIN. Some PBs were stained with monoclonal and polyclonal anti-ubiquitin (see Fig le). The region that was stained with anti-ubiquitin did not always correspond to PB: The cytoplasm around some PBs was also stained, and some PBs were only focally stained (not illustrated). Ubiquitin-positive PBs were most easily observed in the granular cell layer of fascia dentata. They were less frequently observed in the subiculum, cingulate gyrus, and neocortex and very rarely in the pyramidal cell layer of the hippocampus. In addition to PBs, the dendrites and perikarya of some normal-appearing neurons around the PB-bearing cells were stained with both monoclonal and polycional anti-ubiquitin (see Fig le). The perikarya of BNs also stained diffusely with polyclonal and monoclonal anti-ubiquitin (see Fig 2e). The staining intensity was variable and correlated well with the degree of swelling. Some swollen cell processes around BNs also stained with the antibodies (not illustrated). A small percentage of granulovacuolar degenerations (see Fig 2e) and Hirano bodies (not illustrated) were also stained with the antibodies. In the 4 questionable cases of PD, a few neurons were diffusely stained weakly with monoclonal and polyclonal anti-ubiquitin, but these cells did not show ballooning or contain PBs. The antibodies intensely stained the majority of neurofibrillary tangles of the AD type, degenerative cell processes around senile plaques, and neuropil threads in the cases of AD, but weakly stained a small percentage of neurofibrillary tangles in the cases of progressive supranuclear palsy. N o neuronal perikarya stained diffusely in the cases of AD or progressive supranuclear palsy. The antibodies stained diffusely the perikarya of the BNs in a case with pellagra encephalomyelopathy and in a case with Creutzfeldt-Jakob disease. AND A N T I - w 2 . Many PBs were stained intensely with anti-MAP2 (see Fig lc), but,not with anti-tubulin (not illustrated). The perikarya of BNs were diffusely stained with these antibodies, but in some BNs, a focal region was intensely stained with anti-MAP2 (see Fig 2d), but not with anti-tubulin.

ANTI-TUBULIN

Fig I . lmmunocytochemical properties of Pick's argentophilic bodies (PB). (a through d) Pyramidal cell layer of hippocampus. (e) Granular cell hyer dfascia dentata. (a) PB (arrow) with Bodian stain (Case I ) . (6) PBs with monoclonal anti-neurofilament (NF) 200-kDa protein antibody. Negative PBs are surrounded bj positive cytoplasm (arrow) and negative cytoplasm (double arrows) (Case2). (Counterstainedwith hematoxy1in.j (c) PB (arrow) with anti-microtubule-associated protein 2 anti-

body (MAP2) (Case 1}. (Counterstainedwith hematoxylin.) (d) PBs (arrows) within neurons and small structures (arrowheads) within neuropil around PB-bearing cells with anti-phosphorylated tau antibody (PTAU) (Case 1). (e) An anti-gbiquitin (UBQ) polyclonal antibody stains PBs (arrows). In addition, the perikaryal cytoplasm and the dendrite of n o m l appearing neurons are diffusely stained (arrowheads) (Case 7). (Avidin-biotin-complex immunostain, X 700.)

Murayama et ak Immunocytochemistry of Picks Disease 397

Fig 2. Immunocytochemicalproperties of the ballooned neurons (BNs) in Picks disease. (a) BNs with Bodian stain. A focal argentophilic region (arrow) and a vacuole (arrowhead) are present in the perikaryon (Case 2, inferior temporal gyms). (6) The same neuron as in Figure 2a stained with anti-phosphorylated tau antibody (PTAU). AfocaIL$positive region (arrow) of perikaryal cytoplasm is shown. (c) A focally positive region (arTOW)of perikaryal cytoplasm with anti-paired helical filaments antibody (PHF) (Case 1, subiculum). (4 A focally strongly positive region (arrow) within diffusely staining cytoplasm with

anti-microtubule-associated protein 2 antibody (MAP2) (Case I, subiculum). (Counterstained with hematoxylin.) (e) Diffuse& stained cytoplasm with monoclonal anti-ubiquitin (UBQ). A positive granulovacuolar degeneration (arrow) and a negative vacuole (arrowhead) are shwwn (Case 2, parahippocampal gyms). ( f ) Diffusely stained cytoplasm with monoclonal antineurofilamnt 200-kDa protein antibody (NF) (Case2, parahippocampalgyrus). (Counterstained with hematoxylin.) (Avidin-biotin-complex immunostain, x 700.j

Fig 3 . Ultrastructureof Pick's argentophilic body (PB) (Case 2). (a) PB with toluidine blue stain (pyramidal cell layer of hippocampus). ( x 900 before 8% reduction.) (b) Straight filaments, a paired twisted profile (arrow), and a short fragment of paired twisted profile (arrowheads) in PB. A 25-nm tubular structure (microtubule?)(double arrows) is present. Bar = 0.5 em.

(c) Randomly oriented, approximately 15-nm, straight filaments and a short fragment ofpaired twistedprohle (double arrowheads) in a cell process in stratum lacunosum. A membrane thickening (arrow) and a dendrite (0)are also shown. Bar = 5 pm.

Murayama et al: Immunocytochemistry of Pick's Disease 333

Table 2. Immunocytochemical Properties of Pick's Body (PB) and the Ballooned Neuron (BN} in Pick's Disease ~

PB BN

ADNFT PSPNET

Ptau

PHF

++

++

- to

++ ++

++a

- to

++ ++

Ubiquitin ++a

-to +to

++

- to

++ ++

+

NF200K

MAP2

-

-to to

++ -

+

-to -to

++ ++a

++

++

Tubulin -

+ -

"Focally strongly positive.

PB = Pick's body; BN = ballooned neuron; ADNJT = Alzheimer's neurofibrillary tangles; P S P N R = neurofibrillary tangles in progressive supranuclear palsy; ptau = anti-phosphorylated tau antibody; NF200K = monoclonal and polyclonal anti-neurofilament 200-kDa protein antibody; MAP2 = anti-microtubule-associatedprotein 2 antibody; tubulin = anti-tubulin antibody; + + = strongly positive; + = posirive; = negative staining.

The perikarya of the BNs in the disorders other than PD were also diffusely stained with these antibodies, but no focal structures were positive to antiMAP2. ANTI-NF200K. PBs were not recognized by polyclonal and monoclonal anti-NF200K, but the cytoplasm around PBs showed variable staining intensity (see Fig lb). The antibodies intensely stained the perikarya of all BNs (see Fig 2f). They also stained occasional swollen neuronal processes around BNs (not illustrated). in the 4 questionable cases of PD, a few neuronal perikarya were stained moderately with the antibodies, but these positive neurons did not show ballooning. N o neurofibrillary tangles of the AD or progressive supranuclear palsy type were stained with the antibodies. The perikarya of all BNs in the disorders other than PD were also intensely stained with the antibodies. These immunocytochemical results are summarized in Table 2. Electron Microscopy PBS AND NEUROPIL. PBs consisted of an accumulation of randomly oriented straight filaments (see Fig 3b). The diameter of the filaments varied from 12 to 18 nm, with the majority around 15 nm. Paired twisted profiles with minimal diameter of 13 nm, maximal diameter of 26 nm, and twist periodicity of 120 nm (see Fig 3b) were mixed with the straight filaments. Short fragments of the paired twisted profiles were constantly observed in every PB examined (see Fig 3b). Vesicular or granular structures were trapped among the filaments. Twenty-five-nanometer tubular structures (microtubules?) (see Fig 3b) and neurofilaments (not illustrated) were observed in or around PBs. in the perikarya of some normal-appearing neurons, small numbers of approximately 15-nm straight filaments and short fragments of paired twisted profiles were also observed. The neurofilaments were slightly increased in these neurons (not illustrated).

The accumulation of randomly oriented, approximately 15-nm straight filaments and occasional short fragments of paired twisted profiles was also observed in the proximal part of dendrites (not illustrated) and in the cell processes in the neuropil around PB-bearing cells. These PB-type filaments in the neuropil were most easily detected in the stratum lacunosum of the pyramidal cell layer of hippocampus (see Fig 3c). BNS. BNs showed a relative paucity of organelles in the center of the perikaryal cytoplasm. Lipofuscin granules were displaced toward the periphery, and no rough endoplasmic reticulum was observed (see Fig 4b). In higher magnification, BNs were shown to contain sparse membranous and vesicular structures, mitochondria, clumps of granules, and interspersed neurofilaments (see Fig 4c). Fifteen-nanometer straight filaments (see Fig 4d) or short fragments of paired twisted profiles (see Fig 4e) were mixed with the neurofilaments and focally formed small accumulation with random array (not illustrated).

CONTROLS. Straight filaments of 15 nm in diameter were the major components of neurofibrillary tangles in progressive supranuclear palsy and were also observed in neurofibrillary tangles in AD in the hippocampus, where they were mixed with typical paired helical filaments. The 15-nm filaments usually formed bundles and did not form the random meshworks found in PD. Short fragments of paired twisted profiles were never observed among them. The BNs in Creutzfeldt-Jakob disease were ultrastructurally similar to the BNs in PD, except that neither 15-nm straight filaments nor short fragments of paired twisted profiles were observed in the perikarya. lmmunoehctron Microscopy The reaction product with anti-ptau resided on PBs in perikarya (Fig 5a). At higher magnification, randomly oriented filaments were decorated with the antibody (see Fig 5b). Small positive structures that were similar

Murayama et al: Immunocytochemistry of Pick's Disease 401

Fig 5 . lmmunoelectron microscopy of Pick’s argentophilic body (PB) with anti-phosphorykzted tau antibody (PTAU) (Case It frozen sections, the same region as in Figure Id). (Indirectimmunoperoxidzsestain.) (a) The immunoreactiuity with antiptau (arrows) in PB. Bar = 0.5 pm. (b) Higher magnification of randomly orientedjilaments (arrowheads), which are deco-

402 Annals of Neurology Vol 27 No 4 April 1990

rated with the reaction product. Bar = 0.5 pm. (c) Many small foci offilamentous structures in the neuropil decorated with antiptau (arrows). Bar = 0.5 pm. (4 Higher magnification of one of the structures (large arrows). Thick (arrowhead) and thin (small arrow) filaments lie in parallel fashion. Bar = 0.5 pm.

~

to PBs in the perikarya were scattered within the neuropil (see Figs 5c and 5d). In the ubiquitin-positive PBs (Fig 6a), the reaction product with anti-ubiquitin was located in PBs but not in the surrounding cytoplasm (see Fig 6b). In higher magnification, randomly oriented filaments were shown to be decorated with the antibody (see Fig 6c).

Discussion Our study provides new evidence for the following two points. 1. P D shows characteristic neuropil pathology, which is different from either AD or progressive supranuclear palsy. 2. PB-bearing cells and BNs show unique immunocytochemical and ultrastructural properties that may be a clue to the pathogenesis of PD.

1mm.wzocy tochemical and Ultra.rtructural Characterization and Localization of PB-Type Filaments Ultrastructurally PBs consisted of an accumulation of randomly oriented, approximately 15-nm straight filaments 122-251 and paired twisted profiles {26-291. A short fragment of the paired twisted profile was consistently observed in every PB in all 4 cases we examined and was a good marker of PB-associated filaments.

Fig 6. Immunoelectron microscopy of Pick body (PB) with monoclonal anti-ubiquitin (UBQ) (vibratome section, the same region as in Figure le). (Indirect immunoperoxiduse stain.) (a) Positively stained PB. ( x 700 before 9% reduction.) (b) The reaction product in PB. N = nucleus. Bar = 0.5 pm. (c) Higher mugni&ation of randomly orientedJikzmentswhich are decorated. Bar = 0.5 pm.

Our immunocytochemical study confirms the previous reports that certain epitopes [25, 28-31), such as tau 1321 are shared by PBs and neurofibrillary tangles of the AD and progressive supranuclear palsy type. Immunocytochemical study with anti-ptau showed that small ptau-positive filamentous structures were scattered in the neuropil around PB-bearing neurons. The changes in neuropil were hard to visualize with routine silver staining. Ultrastructurally, some of these structures apparently correspond to PB-type filaments in neuronal processes. The pathological changes in the neuropil have been stressed in AD [33), but not fully documented in PD. Our study demonstrates for the first time that PB-type filaments also occur in neuronal processes. An epitope of ptau was detected in small amounts in BNs, which corresponds to small focal areas of argyrophilia. Ultrastructurally, PB-type filaments were observed in small amounts in BNs. These histological, immunocytochemical, and ultrastructural properties were unique to the BNs in PD and quite distinct from the BNs in disorders other than PD. Murayama et al: Immunocytochemistry of Pick's Disease 403

The Sign&cance of Ubiquitin in the Pathogenesis of PD Some PBs, but not all, were stained with anti-ubiquitin 132, 341. The region that was stained with antiubiquitin did not always correspond to PB 134). Moreover, the perikarya and dendrites of some normalappearing neurons around the PB-bearing cells were stained with the antibodies. These immunocytochemical results with anti-ubiquitin were quite distinct from AD or progressive supranuclear palsy. In AD, intense ubiquitin reactivity was observed in almost all neurofibrillary tangles, degenerated cell processes around senile plaques, and neuropil threads 115, 351, while in progressive supranuclear palsy, only rare ubiquitinpositive neurofibrillary tangles were observed 1321. Since ubiquitin has a physiological role by serving as an initial step in ATP-dependent nonlysozomal proteolysis 1151, we speculate that the diffuse staining of neuronal perikarya and dendrites indicates an early stage of cell injury that may lead to the formation of PB-type filaments. To identify the proteins conjugated by ubiquitin, the immunoelectron microscopic study of these diffusely stained neurons was carried out. Unfortunately, the reaction product was hard to localize to any definite structures. We also tried to identify biochemically the proteins conjugated by ubiquitin. The granular cell layer of fascia dentata was trimmed from frozen sections of the hippocampus of Case 3 and processed for immunoblotting with the anti-ubiquitin monoclonal antibody [lS}, but no band was identified, probably owing to a low sensitivity. Further immunocytochernical and biochemical studies should address this issue. The perikarya of the BNs were stained diffusely with the anti-ubiquitin antibodies. Our neurofilament antibodies, which did not recognize neurofibrillary tangles of the AD type, stained the perikarya of all BNs intensely. The antibodies did not stain PBs 128, 36, 371, but intensely stained some perikarya around PBs 1371. Several studies showed that PBs and BNs share the epitope of neurofilament 1361, and that this epitope is phosphorylated 138, 391. However, because these antibodies possibly cross-react with tau proteins 1401, further studies are needed. Ballooned neurons is a descriptive term based on routine histological stainings. BNs are thought to be common in various disorders 1411, although recent immunocytochemical and ultrastructural studies show that BNs are different in each disorder C38,42, 431. The ultrastructural studies of the B N in PD are very few 144-463 and mutually conflicting. Our studies demonstrate that the BNs in P D contain PB-type filaments and the epitope of ptau and ubiquitin and may represent a distinctive pathological finding in PD. Because BNs, as well as PBs, may be a clue to the pathogenesis of PD, further immunocytochemical and ultrastructural studies of BNs are indicated. 404 Annals of Neurology Vol 27 No 4 April 1990

Supported in part by Grants in Aid from the Ministry of Education, Science and Culture, Japan, and the Sasakawa Foundation. We thank Drs R. Takanashi (Case l), K. Inoue (Cases 2 and 7), K. Arima (Cases 3, 4, 9, 11, and 12), K. Kosaka (Case 5). S. Kato (Cases 8 and 14), Y. Nakazato (Case 10) and the late Y. Urano (Case 13) for permitting us to examine the individual cases; Dr A. L. Haas for providing us with the affinity-purified antibody to ubiquitin; Dr H. Yamaguchi for providing us with the antibody to the 200-kDa human neurofilament; and Dr N. Nukina for providing us with the anti-MAP2 antisera. We also thank Dr K. Suzuki for helpful comments on the immunocytochemical and ultrastructural studies, and Drs D. L. McIlwain and T. W. Bouldin for discussion and editing of the manuscript. Ms J. Saishoji, T. Hayashida, and C. Iangaman helped us with technical support. Mrs D. Sears prepared the manuscript.

References 1. Pick A. Ueber die Beziehungen der senilen Hirnanophie zur Aphasie. Prag med Wochennschr 1892;17:165-167 2. Onari K, Spatz H. Anatomische Beitraege zur Lehre von der Pickschen umschriebenen Grosshirnrinden-Atrophie (“Picksche Krankheit”) Z Neurol Psychiatr 1926;101:470-511 3. Lueers T, Spatt H. Picksche Krankheit (progressive umschdebene Grosshirnatrophie). In: Lubarsch 0,Henke F, Roessle R, eds. Handbuch der speziellen pathologischen Anatomie und Histologie, Bd 13. Berlin: Springer, 1957:614-715 4. Altheimer A. Ueber eigenartige Krankheitsfaelle des spaeteren Alters. 2 Neurol Psychiatr 1911;4:356-385 5. Spielmeyer W. Eigenartige organische Psychosen. In: Aschaffenburg G, ed. Handbuch der Psychiatrie, Spezieller Teil, 5 Abteilung. Leipzig: Franz Deuticke, 1912:141-146 6. Rtchter H . Eine besondere Art von Stirnhirnschwund mit Verbloedung. 2 Neurol Psychiatr 1918;38:127-160 7. Ferraro A, Jervis G. Pick‘s disease. Clinicopathologic study with report of two cases. Arch Neurol Psychiatry 1936; 36:739-767 8. Williams HW. The peculiar cells of Picks disease. Arch Neurol Psychiatry 1935;34:508-519 9. Schneider C. Ueber Picksche Krankheit. Monatsschr Psychiatr Neurol 1927;65:230-27 5 10. Constantinidis J, Richard J, Tissot R. Pick‘s disease. Histological and clinical correlations. Eur Neurol 1974;11:208-2 17 11. Newman MA. Pick‘s disease. J Neuropathol Exp Neuroi 1949;8:255-282 12. Seitelberger F, Gross H, Pilz P. Picks disease: a neuropathological study. In: Hirano A, Miyoshi K, eds. Neuropsychiatric disorders in the elderly. New York: Igaku-Shoin, 1983537-117 13. Ihara Y, Nukina N, Miura R, Ogawara M. Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer’s disease. J Biochem 1986;99:1807-1811 14. Ihara Y, Carmela A, Selkoe DJ. Antibodies to paired helical filaments in Alzheimer’s disease do not recognize normal brain proteins. Nature 1983;304:727-7 30 15. Mori H , Kondo J, Ihara Y. Ubiquitin is a component of paired helical filaments in Alzheimer’s disease. Science 1987;235: 1641- 1644 16. Haas AL, Bright PM. The immunochemical detection and quantitation of intracellular ubiquitin-protein conjugates. J Biol C hem 1985;260:12464- 12473 17. Yamaguchi H, Hirai S, Tanaka M, Shooji M. Basic studies on the application of anti-human neurofilament antibody on formalin-fixed paraffin-embedded tissue sections. NeuropatholOgy (Kyoto) 1984;5:377-384 18. Nukina N, Ihara Y. Immunocytochemical study on senile plaques in Alzheimer’s disease. 1. Preparation of antimicrotu-

bule-associated proteins (MAPS) and its specificity. Proc Jpn Acad 1983;59B:284-287 19. Ihara Y, Nukina N, Sugita H, Toyokura Y.Staining of Alzheimer's neurofibrillary tangles with antiserum against 200k component of neurofilament. Proc Jpn Acad 1981;57B:152156 20. Kuzuhara S, Mori H , Izumiyama N, et al. Lewy bodies are ubiquitinated. A light and electron microscopic immunocytochemical study. Acta Neuropathol (Berl) 1988;75:345-353 21. Hsu SM, Raine L, Fanger H. A comparative study of the peroxidase-antiperoxidase method and an avidin-biotin complex method for studying polypeptide hormones with radioimmunoassay antibodies. Am J Clin Pathol 1981;75:734-738 22. Schochet S, Lampert PW, Lmdenberg R. Fine structure of the Pick and Hirano bodies in a case of Pick's disease. Acta Neuropathol (Berl) 1968;11:330-337 23. Towfighi J. Early Pick's disease. A light and ultrastructural study. Acta Neuropathol (Berl) 1972;2 1:224-231 24. Oyanagi S. Ultrastructural comparison between Pick bodies and the neurofibrillary changes in Alzheimer's disease and progressive supranuclear palsy. In: Hirano A, Miyoshi K, eds. Neuropsychiatric disorders in the elderly. New York Igaku-Shoin, 1983:118-126 25. Perry G, Stewart D, Friedman R, et al. Filaments of Pick's bodies contain altered cytoskeletal elements. Am J Pathol 1987;127:559-568 26. Shibayama H, Kitoh J, Marui Y, et al. An unusual case of Pick's disease. Acta Neuropathol (Berl) 1983;59:79-87 27. Munoz-Garcia D, Ludwin SK. Classic and generalized variants of Pick's disease: a clinico-pathological, ultrastructural, and immunocytochemical comparative study. Ann Neurol 1984;16: 467-480 28. Rasool CG, Selkoe DJ. Sharing of specific antigens by degenerating neurons in Pick's disease and Alzheimer's disease. N Engl J Med 1985;312:700-705 29. Yen S-H, Dickson DW, Peterson C, et al. Cytoskeletal abnormalities in neuropathology. Prog NemJpathol 1986;6:6390 30. Pollock N, Mirra SS, Binder LI, et al. Filamentous aggregates in Pick's disease, progressive supranuclear palsy, and Alzheimer's disease share antigenic determinants with microtubule-associated protein, tau. Lancet 1986;1:1211 3 1. Murayama S, Nukina N , Ihara Y, et al. Immunocytochemistry of Pick's argentophilic bodies: evidence for the involvement of high molecular weight microtubule associated proteins (HMWP) before the appearance of tubulin. Rinsho Shinkeigaku 1985;25:80-87 32. Love S, Saitoh T, Quijada S, et al. Alz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies. J Neuropathol Exp Neurol 1988;47:393-405

33. Kowall NW, Kosik K. Axonal disruption and aberrant localization of tau protein characterize the neuropil pathology of Alzheimer's disease. Ann Neurol 1987;22:639-643 34. Lowe J, Blanchard A, Morrell K, et al. Ubiquitin is a common factor in intermediated filament inclusion bodies of diverse type in man, including those of Parkinson's disease, Pick's disease, and Alzheimer's disease, as well as Rosenthal fibers in cerebellar astrocytomas, cytoplasmic bodies in muscle, and Mallory bodies in alcoholic liver disease. J Pathol 1988;155:9-15 35. Manetto V, Perry G, Tabaton M, et al. Ubiquitin is associated with abnormal cytoplasmic filaments characteristic of neurodegenerative diseases. Proc Natl Acad Sci USA 1988;85: 4501-4505 36. Probst A, Anderton BH, Ulrich J, et al. Pick's disease: an immunocytochemical study of neuronal changes. Monoclonal antibodies show that Pick bodies share antigenic determinants with neurofibrillary tangles and neurofilaments. Acta Neuropathol (Berl) 1983;60: 175- 182 37. Nakazato Y, Sasaki A, Hirato J, et al. Immunohistochemical localization of neurofilament protein in neuronal degenerations. Acta Neuropathol (Berl) 1984;64:30-36 38. Dickson DW, Yen SH, Suzuki KI, et al. Ballooned neurons in select neurodegenerative diseases contain phosphorylated neurofilament epitopes. Acta Neuropathol (Berl) 1986;7 1:216223 39. Ulrich J, Haugh M, Anderton BH, et al. Alzheimer dementia and Pick's disease: neurofibrillary tangles and Pick bodies are associated with identical phosphorylated neurotilamenr epitopes. Acta Neuropathol (Berl) 1987;73:240-246 40. Nukina N, Kosik KS, Selkoe DJ. Recognition of Altheimer paired helical filaments by monoclonal neurofilament antibodies is due to cross reaction with tau protein. Proc Natl Acad Sci USA 1987;84:3415-3419 41. Clark AW, Manz HJ, White CL 111, et al. Cortical degeneration with swollen chromatolytic neurons: its relationship to Pick's disease. J Neuropathol Exp Neurol 1986;45:268-284 42. Murayama S , Mori H, Ihara Y, et al. Immunocytochemical and ultrastructural study of lower motor neurons in amyotrophic lateral sclerosis. Ann Neurol 1990;27:137-148 43. Murayama S, Bouldin TW, Suzuki K. Immunocytochemical and ultrastructural studies of Werdnig-Hoffman disease. Ann Neurol 1989;26:303 (Abstract) 44. Wisniewsky HM, Coblentz JM, and Terry RD. Pick's disease. A clinical and ultrastructural study. Arch Neurol 1972;26:97-108 45. Brion S, Mikol J, Psimaras A. Recent findings in Pick's disease. Prog Neuropathol 1973;2:421-452 46. Arima K, Miyasaka Y. An autopsy case of Pick's disease, with special reference to light and electron microscopical findings of Pick bodies and ballooned neurons. Neuropathology (Kyoto) 1985;6:325-339

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