386
585 (1992) 386-390 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05,00
BRES 25241
Lewy bodies contain beta-amyloid precursor proteins of Alzheimer's disease Hiroyuki Arai *, Virginia M.-Y. Lee, William D. Hill **, Barry D. G r e e n b e r g *** and J o h n Q. Trojanowski Department of Pathology and Laboratory Medicine, Dit,ision of Anatomic Pathology, The University of Pennsyh,ania School of Medicine, Philadelphia, PA 19104 (USA) (Accepted 24 March 1992)
K~' words: Lewy body; Alzheimer's disease; Parkinson's disease;/3-Amyloid precursor protein
To assess the contribution of Alzheimer's disease amyloid proteins to cortical and substantia nigra Lewy bodies (LBs), regions of postmortem brain rich in intraneuronal LBs were examined immunohistochemically. Antibodies to epitopes in domains outside the amyloidogenic beta-amyloid peptide (BAP) in BAP precursor proteins (BAPPs) as well as to the BAP itself were used as probes. These studies showed that only BAPP epitopes outside the BAP were present in substantia nigral and cortical LBs. Thus, non-amyloidogenic domains of BAPPs may be associated with intraneuronal inclusions comprised of neurofilament proteins.
Lewy bodies (LBs), a pathological hallmark of Parkinson's disease (PD), are eosinophilic cytoplasmic inclusions that occur in neurons of selected brainstem nuclei including the substantia nigra (SN), locus ceruleus, and the dorsal vagal motor nucleus, They also occur in selected telencephalic nuclei (e.g. substantia innominata) and in cortical neurons (for review see Forno~). lmmunohistochemical studies have shown that LBs label with antibodies to neurofilament (NF) triplet proteins~,t0-~2,1s,lsj0, uhiquitin4,0.ts.23.aT,~0 tubulin and high molecular weight microtubule-associated proteins (e.g. MAPs la and 2)4.t~,2~, but not with antibodies to tau 's'12.lS'z3'zTJ°.Although the uitrastructure of LBs is dominated by aggregated filaments that appear to be derived, at least in part, from NF proteins, LBs have yet to be isolated, purified and subjected to biochemical analysis. Hence, it has been necessary to employ immunohistochemical methods to dissect the composition of LBs. The idea that Alzheimer's disease (AD) and PD may overlap has been debated for a number of years, It
has been argued that AD and PD may share some common pathogenic mechanisms, or that the presence of one disease may predispose an individual to acquire the second4,'j,lq,2~','~°,~s,and even that AD and diffuse Lewy body disease (DLBD) belong to the same spec. trum of disease processes~,1~,Some studies suggest that concomitant AD and PD pathologies occur with a frequency greater than chance alone predicts s,7,t6`2°,2o, while others have suggested that these co-occurrences are statistically non.significant t4, Many, but not all, PD subjects presenting with clinical signs of dementia exhibit cortical pathology typical of AD ts't4't~,3s, and subcortical as well as cortical LBs also occur in a percentage of AD patients 6,~,t4,t~,:~,2s, While the co-existence of AD and PD pathologies in the same patients is not uncommon, the significance of this association is not well understood, Only recently has it been shown that senile plaques (SPs) in the hippocampus of a subset of demented PD subjects exhibit the same molecular features as those found in SPs of the AD hippocampus -~, rather than the features found in the SPs of neurologi-
* Present address: Department of Neurology, Motojima General Hospital, eta, Gunma 373, Japan. ** Present address: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912-2000, USA. *** Present address: The Upjohn Company, Kalamazoo, MI 49001, USA. Correspondence: J.Q. Trojanowski, Department of Pathology and Laboratory Medicine, Division of Anatomic Pathology, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
387 TABLE I
;;'ammary of antibody probes This table summarizes the immunochemical and immunohistochemical properties of the antibodies used in this study, aa, amino acid; BAP, beta-amyloid peptide; BAPP, beta-amyloid precursor protien; COOH, carboxT; LBs, Lewy bodies; mAbs, monoclonai antibodies; NFTs, neurofibrillary tangles; SPs, senile plaques.
Antibody
Specificity
LN21, 27, 39
These 3 mAbs are to epitopes within the first 100aa of BAPPs 695, 751 and 770; they label neurons, glia, extracellular NFTs, LBs, and the corona of SPs Antisera to aa 18-35 of BAPP 695; it labels the corona of SPs Antisera to aa 301-319 of BAPP 695 (glycosylation site); it labels the corona of SPs Antisera to va 1-42 of BAP; it labels NFTs, diffuse plaques and SPs mAb to aa 1-28 of BAP; it labels NFTs, diffuse plaques and SPs mAb to NF-M phosphorylation dependent COOH tail domain; it labels LBs but not NFTs Anti-ubiquitin mAb; it labels LBs, NFTs, neuropil threads, and SPs
Up84
ups6 UpI07 Amy33 RMO32 MABI510
cally normal controls. Further, the cortical LBs found in some AD patients contain the same constellation of NF protein epitopes that are found in cortical and SN LBs in PD patients Is'3°. This suggests that both SPs and LBs in AD and PD may result from linked pathological events. To pursue this issue further we probed cortical ~nd SN tissue samples from PD, AD/PD, and DLBD patients, as well as from neurologically normal control cases using antibodies to the beta-amyloid peptide (BAP) and other BAP precursor (BAPP) epitopes to determine if the BAP or its precursors were contained in the LBs found in these tissues. Monoclonal antibodies (mAbs) to the recombinant form of the full-length 695 a~i:lo acid (aa) BAPP were characterized and described elsewhere ~'a. These included 3 well.characterized mAbs (LN21, LN27 and LN39) that recognize epitopes within the first 100 aa of
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,.
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the amino-terminus of all three major BAPPs (BAPP695, BAPP751, and BAPP770). None of these mAbs cross-react with *;,,*ergent-insoluble cytoskeletal proteins derived from h,,nan brain. Additional antibodies were used that were raised to synthetic peptide homologs of different BAPP domains. These included: rabbit antisera Up84 (raised to aa 18-35 of BAPP695), Up86 (raised to aa 301-319 of BAPP695), and Upl07 (raised to aa 1-42 of BAP) ~. A second probe for BAP was also used, the mAb AMY33 (raised to aa 1-28 BAP) 3a. Another mAb, RMO32, which recognizes a phosphate-dependent epitope in the tail region of the middle molecular weight NF protein (NF-M) ms's°, was used since it specifically detects LBs and does not recognize neurofibrillary tangles (NFTs). Finally, a commercially available anti-ubiquitin mAb (Chemicon MAB 1510) which labels NFTs and LBs was employed
P .,
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Fig. 1. a: two LBs in the substantia nigra pars compacta are labeled by LN21. Note that the peripheries of the LBs are intensely stained (indicated by arrows), b: a cortical LB (parahippocampal gyrus) is shown here labeled with LN21 (arrow head)• In contrast to substantia nigra LBs those in the cortex are homogeneously labeled by the LN mAbs; note the uniform staining of the LB. c: the two LBs shown here (arrows) are unlabeled by UP107. The sections in these photomicrographs were counterstained with hematoxylin. Scale bars ffi 10 ~m.
388 as well is'3°''~. The properties of these antibodies are summarized in Table I. Brains were collected at autopsy from 14 PD cases (i.e. 4 non-demented PD patients, 5 demented PD patients without AD pathology, and 5 demented PD patients with AD pathology), 3 A D / P D cases (i.e. demented patients with a clinical diagnosis of AD and postmortem findings of AD and PD pathology), and 10 control patients without AD, PD or other neurodegenerative diseases. One case of DLBD was also studied. The clinical data and quantitative epitope analysis of hippocampal SPs in these patients has been described in detail elsewhere 3. The brains were either fixed in 10% neutral-buffered formalin, Bouin's solution, 70% ethanol/150 mM NaCI, or denatured by microwave energy. Both the fixation protocols and the performance of the antibodies used here with tissues denatured in these fixatives have been previously described ~-3.~s.3°.32'33. The tissue blocks were embedded in paraffin, sectioned at 6 ~tm, and then probed with the different antibodies using nearly consecutive sections according to previously published immunoperoxidase procedures ~-3'18'24'25'3°'32'33.Some sections were pretreated with 90% formic acid for 5 min prior to immunostaining to improve detection of BAP 2'32. The diagnosis of AD was established using the criteria recommended by the National Institute on Aging 2t, while PD and DLBD were diagnosed as described earlier*.tH.30. As reported previously 1°3, the anti-amino.terminal BAPP mAbs LN21. LN27, and LN39 stained the corona of SPs, neuronal cell bodies, neuronal processes, gila) and extracellular NFTs. Additionally, all three mAbs stained both SN and cortical LBs. Notably, in 'typical' SN LBs, the periphery of the LB core was intensely labeled, while the central core and halo of these LBs appeared unstained at the light microscopic level (Fig.
la). The BAPP immunoreactivity was present within SN LBs of all 14 PD patients, the 3 AD cases that had concomitant PD pathology in the SN, and the DLBD case. RMO32 and the anti-ubiquitin mAb labeled these LBs in adjacent sections. Corti¢ai LBs exhibited homogeneous or slightly granular staining with LN21, LN27 and LN39 (Fig. lb). immunolabeled cortical LBs were found in all 14 PD patients, the 3 AD patients with PD neuropathology, and the DLBD patient. Similar staining was not seen in the brains of the 10 control cases, all of which lacked SN and cortical LBs (as defined by H& E, RMO32, and anti-ubiquitin staining). Cortical and SN LBs were negative when probed with the anti-BAP Abs Upl07 and AMY33, even after pretreatment of the sections with formic acid. in contrast,
diffuse amyloid plaques, SPs and NFTs were stained with Upl07 and AMY33 on these same sections. This suggests that the amyloidogenic BAP does not accumulate within LBs (Fig. lc). The cortical LBs were distinguishable from tangleladen neurons, dystrophic processes in the neuropil and SPs using RMO32 3'~s'3°. Furthermore, only a subset of LBs appear to contain BAPP epitopes, since fewer LBs were labeled by LN21, LN27 and LN39 than with RMO32 and the anti-ubiquitin mAb. Relative to RMO32, approximately 30-40% of SN LBs and 1020% of cortical LBs were detected with these BAPP mAbs. However, this is consistent with the immunodetection of other proteins in LBs, including many, if not most, NF protein epitopes ~s'a°. This phenomenon may reflect post-translational modifications of NF and BAPP proteins affecting their tertiary conformation and epitope antigenicity. Additionally, LBs which lack NF and BAPP immunoreactivity may contain the epitopes for which they are being probed, but they may be below the threshold level required for detection with these specific antibodies. Alternatively, the association of BAPP with LBs may occur secondary to LB formation. In contrast to the LN series of mAbs, no convincing immunostaining was obtained with either Up84 or Up86. However, it has been noted previously that these polyclonal antibodies are less sensitive than the LN mAbs in recognizing BAPPs 1'3. Thus, relatively low levels of BAPPs in LBs may go undetected with these antibodies. This is the first study to describe the presence of BAPPs in cortical and SN LBs. However, it is not clear if our data imply that amino-terminal fragments or full-length BAPPs are present in these LBs. If BAPPs play a role in the formation of LBs, it could be a result of the ability of BAPPs to bind to and interact with cytoskeletal proteins. This possibiliW is raised by the observation that BAPPs partition with detergent-insoluble cytoskeletal preparations 2~. Whether BAPPs interact directly with cytoskeletal elements or through other proteins is not clear, it is also possible that the findings reported here represent the interaction of BAPP-containing vesicles with the cytoskeleton. Since Refolo et al. 2~ have provided evidence that BAPP/cytoskeleton interactions are microtubule-dependent, and the full-length BAPPs are integral membrane proteins, it is possible that BAPPs reside in cytoplasmic vesicles as integral membrane proteins that interact with the cytoskeleton. Duffy and Tennyson s have shown that large numbers of small vesicles are localized to the periphery of the central core of SN LBs. Thus the distribution of BAPP immunoreactivity in SN LBs seen in this study may reflect the accumulation of BAPP
389
containing-vesicles within LBs. If LBs are in part formed through the disturbance or collapse of the neuronal cytoskeleton, then vesicles with membranes rich in BAPP may be associated with one or more of the cytoskeletal components that form the filamentous meshwork of the LB. That a cytoskeletal disturbance could be initiated by one or more of the BAPPs or the amyloidogenic BAP itself is suggested by studies of BAPs injected into the brains of rats which showed alterations in tau protein immunoreactivity 22. Further, BAP immunoreactivity is frequently associated with N F T s I-3. Therefore, disturbances in BAPP expression or metabolism may trigger pathogenic event~ involving the neuronal cytoskeleton. Recently, the amyloidogenic protein found in familial amyloidosis, Finnish-type (FAF), has been partially sequenced and found to be identical to a portion of gelsolin except for a single aa substitution 34. An antiserum raised to the purified FAF amyloid was found to label SN and cortical LBs in PD and DLBD. The finding of gelsolin, an actin binding and regulatory protein, or a gelsolin-related molecule in LBs is additional evidence that the LB may represent a general disturbance or collapse of the neuronal cytoskeleton. Further, the identification here of BAPPs in LBs, together with the data on FAF, show that two distinctly different amyloidogenic peptides accumulate in LBs. This constitutes additional evidence linking disruption of the neuronal cytoskeleton and amyloidogenesis in neurodegenerative diseases. The authors wish to thank Mr. G, DiDario l'~r help with the figures and Ors, G. GottUeb, M. Grossman, R.C. Gur, !1.1. Hurtig, as well as Ms, M, Phoenix R,N., and the residents and staff of the Division of Anatomic Pathology who aided in the collection of postmortem tissues, We wish to add special thanks to the patients and their families who made our work possible through their generous efforts to foster research, and the Philadelphia Alzheimer's Disease and Related Disorder Association Inc. This work was supported by Nll+I Grants AG-09215 (JOT), AGI0124 (JOT) and AG.05465 (WDH). 1 Arai, H., Lee, V.M.-Y., Messinger, M.L., Greenberg, B.D., Lowery, D.E. and Trojanowski, J.O., Expression patterns of betaamyloid protein (beta-APP) in neural and non-neural human tissues from Alzheimer' s disease and control subjects, Ann. Neurol., 30 (1991) 686-693. 2 Arai, H., Lee, V.M.-Y., Otvos Jr., L., Greenberg, B.D., Lowe~, D.E., Sharma, S.K., Schmid'~ M.L. and Trojanowski, J.O., Defined neurofilament, tau and beta.amyloid protein epitopes distinguish Alzheimer from non..Alzheimer senile plaques, Prec. Natl. Acad. Sci. USA, 87 (1990) 2249-2253. 3 Arai, H., Schmidt, M.L., Lee, V.M.-Y., Hurtig, H.I., Greenberg, B.D., Adler, C. and Trojanowski, J.Q., Epitope analysis of senile plaque components in the hippocampus of patients with Parkinson's disease, Neurology, in press. 4 Bancher, C., Lassmann, H., Budka, t!., Jellinger, K., Grundkelqbal, I., lqbal, K., Wiche, G., Seitelberger, F. and Wisniewski, H.M., An antigenic profile of Lewy bodies: immunocytochemical indication for protein phosphorylation and ubiquitination, J. Neuropathol. Exp. Neurol., 48 (1989) 81-93. 5 Boiler, F., Mizutani, T., Roessmann, U. and Gambetti, P.,
Parkinson's disease, dementia, and Alzheimer disease: clinicopathological correlations, Ann. Neurol., 7 (1980) 329-335. 6 Dickson, D.W., Crystal, H., Mattiace, UA., Kress, Y., Schwagerl, A. and Ksiezak-Reding, H., Diffuse Lewy body disease: light and electron microscopic immunocytochemistry of senile plaques, Acta Neuropathol. (Berlin), 78 (1989) 572-584. 7 Ditter, S.M. and Mirra, S.S., Parkinson's disease in AIzheimer's disease: a neuropathologic and clinical study, Neurology, 36 (suppl.) (1986) 225. 8 Duffy, P.E. and Tennyson, V.M., Phase and electron microscopic observations of Lewy bodies and melanin granules in the substantita nigra and locus coeruleus in Parkinson's disease, J. Neuropathol. Exp. Neurol., 24 (1965) 398-414. 9 Forno, L.S., The neuropatholoay of Parkinson's disease (The Lewy body as a clue to the nerve cell degeneration). In F. I-lefti and W.J. Weiner (Eds.), Progress in Parkinzon Research, Plenum Press, New York, 1988, pp. 11-21. 10 Forno, L.S., Sternberger, L.A., Sternberger, N.H., Strefling, A.M., Swanson, K. and Eng, L.F., Reaction of Lewy bodies with antibodies to phosphorylated and non-phosphorylated neurofilaments, Neurosci. Lett., 64 (1986) 253-258. ! 1 Forno, L.S., Strefling, A.M., Sternberger, L.A., Sternberger, N.H. and Eng, L.F., lmmunocytochemical staining of neurofibrillary tangles and of the periphery of Lewy bodies with a monoclonal antibody to neurofilaments, J. Neuropathol. Exp. Neurol., 42 (1983) 342. 12 Galloway, P.G., Grundke-lqbal, l., lqbai, K. and Perry, G., Lewy bodies contain epitopes both shared and distinct from AIzheimer neurofibrillary tangles, J. Neuropathol. Exp. NeuroL, 47 (1988) 654-663. 13 Gaspar, P. and Gray, F., Dementia in idiopathic Parkinson's disease, Acta Neuropathol. (Berlin), 64 (1984) 43-52. 14 Gibb, W.R.G., Mountjoy, C.Q., Mann, D.M.A. and Lees, A.J., A pathological study of the association between Lewy body disease and AIzheimer's disease, J. Neurol. Neurosurg. Psych., 52 (1989) 701-708. 15 Goldman, J.E., Yen, S.-H., Chiu, F.-C. and Peress, N.S., Lewy bodies of Parkinson's disease contain neurofilament antigens, Science, 221 (1983) 1082-1084. 16 Hakim, A.M. and Mathieson, G., Dementia in Parkinson disease: a neuropathological study, Natt#~,, 29 (1979) 1209-1214. 17 Hansen, L., Salomon, D., Galasko, D., Masliah, E., Katzman, R., DeTeresa, R., Thai, L., Pay, M.M., Hofstelter, R., Klauber, M., Rice, V., Butters, N. and Alford, M., The Lew? body variant of AIzhuimer's disease: a clinical and pathological entity, Neurology, 40 (1990) I-8. 18 Hill, W.D., Lee, V.M.-Y., Hurtig, H., Murray, J.M. and Tro. janowski, J.Q., Epitopes located in spatially separate domains of each neurofilame,t subunit are present in Parkinson's disease Lew? bodies, J. Comp. NeuroL, 309 (1991) 150-160. 19 Jellinger, K., Neuropathological substrates of AIzheimer's disease and Parkinson's disease, J. Neural Transm., 24, Suppl. (1987)
i 09-129. 20 Joachim, C.L., Morris, J.H. and Selkoe, D.J., Clinically diagnosed AIzheimer's disease: autopsy results in 150 cases, Arm. NeuroL, 24 (1988) 50-56. 21 Khacbaturian, Z.S., Diagnosis of Alzheimer's disease, Arch. Net+. rol,, 42 (1985) 1097-1105. 22 Kowall, N.W., Beal, M.F., Busciglio, J., Duff'y, L.K. and Yankner, B.A., An in rive model for the neurodegenerative effecls of/3 amyloid and protection by substance P, Prec. NatL Acad. Sci. USA, 88 (1991) 7247-7251. 23 Kuzuhara, S., Mori, H., Izumiyama, N., Yoshimura, M. and lhara, Y., Lewy bodies are ubiquitinated, Acta Neuropathol. (Berlin), 75 (1988) 345-353. 24 Lee, V.M.-Y., Carden, M.J., Schlaepfer, W.W. and Trojanowski, J.Q., Monoclonal antibodies distinguish several differentially phosphorylated states of the largest rat neurofilament subunits (NF-H and NF-M) and demonstrate their existence in the normal nervous system of adult rats, J. Neurosci., 7 (1987) 3474-3488. 25 Lee, V.M.-Y., Otvos Jr., L., Schmidt, M.L. and Trojanowski, J.Q., AIzheimer disease tangles share immunological similarities with
39O
26 27
28 29 30
31
multiphosphorylation repeats in the two large neurofilament pro. teins` ~ Natl. Acad. Sci. USA, 85 (1988) 7384-7388. Leverenz, J. and Sumi, M., Parkinson's disease in patients with Alzheimer's disease, Arch. Neurol., 43 (1986) 662-664. Love, S., Saitoh, T., Quijada, S., Cole, G.M. and Terry, R.D., AIz-50, ubiquitin and tau immunoreactivity of neurofibrillary tangles, Pick bodies and Lewy bodies, J. Neuropathoi. Exp. New to/., 47 (1988) 393-405. Morris, J., Drazner, M. and Fulling, K., Clinical and pathological aspects of Parkinsonism in Alzheimer's disease: a role for extranigral factors? Arch. Neurol., 46 (1989) 651-657. Refolo, L.M., Wittenberg, I.S., Friedrich, V.L. and Robakis` N., The Alzheimer amyloid precursor is associated with the detergent.insoluble cytoskeleton, J. Neurosci., 11 G991) 3888-3897. Schmidt, M.L., Murray, J.M., Lee, V.M.-Y., Hill, W.D., Wertkin, A. and Trojanowski, J.Q., Epitope map of neurofilament protein domains on conical and peripheral nervous system Lewy bodies, Am. J. Pathol., 139 (1991) 53-65. Shaw, G. and Chau, V., Ubiquitin and microtubule-associated protein tau immunoreactivity each define distinct structures with
32
33
34
35
differing distributions and solubility properties in Alzheimer brain, Pro,:. Natl. Acad. Sci. USA, 85 (1988) 2854-2858. Stern, R.A., Otvos Jr., L., Trojanowski, J.Q., Hill, W.D. and Lee, V.M.-Y., Monoclonal antibodies to a synthetic peptide homologous with the first 28 amino acids of Alzheimer's disease betaprotein recognize amyloid and diverse glial and neuronal cell types in the central nervous system, Am. J. Pathol., 134 (1989) 973-978. Trojanowski, J.Q., Schmidt, M.L., Otvos Jr., L., Arai, H. and Lee, V.M.-Y., Vulnerability of the neuronal cytoskeleton in aging and Alzheimer's disease: widespread involvement of all three major filament systems, Annu. Rev. Gerontol. Geriatrics, 10 (1990) 167182. Wisniewski, T., Haltia, M., Ghiso, J. and Frangione, B., Lewy bodies are immunoreactive with antibodies raised to gelsolin related amyloid-Finnish type, Am. J. Pathol., 138 (1991) 10771083. Yoshimura, M., Pathological basis for dementia in elderly patients with idiopathic Parkinson's disease, Eur. Neurol. (Suppl. 1), 28 (1988) 29-35.
585 (1992) 386-390 © 1992 Elsevier Science Publishers B.V. All rights reserved 0006-8993/92/$05,00
BRES 25241
Lewy bodies contain beta-amyloid precursor proteins of Alzheimer's disease Hiroyuki Arai *, Virginia M.-Y. Lee, William D. Hill **, Barry D. G r e e n b e r g *** and J o h n Q. Trojanowski Department of Pathology and Laboratory Medicine, Dit,ision of Anatomic Pathology, The University of Pennsyh,ania School of Medicine, Philadelphia, PA 19104 (USA) (Accepted 24 March 1992)
K~' words: Lewy body; Alzheimer's disease; Parkinson's disease;/3-Amyloid precursor protein
To assess the contribution of Alzheimer's disease amyloid proteins to cortical and substantia nigra Lewy bodies (LBs), regions of postmortem brain rich in intraneuronal LBs were examined immunohistochemically. Antibodies to epitopes in domains outside the amyloidogenic beta-amyloid peptide (BAP) in BAP precursor proteins (BAPPs) as well as to the BAP itself were used as probes. These studies showed that only BAPP epitopes outside the BAP were present in substantia nigral and cortical LBs. Thus, non-amyloidogenic domains of BAPPs may be associated with intraneuronal inclusions comprised of neurofilament proteins.
Lewy bodies (LBs), a pathological hallmark of Parkinson's disease (PD), are eosinophilic cytoplasmic inclusions that occur in neurons of selected brainstem nuclei including the substantia nigra (SN), locus ceruleus, and the dorsal vagal motor nucleus, They also occur in selected telencephalic nuclei (e.g. substantia innominata) and in cortical neurons (for review see Forno~). lmmunohistochemical studies have shown that LBs label with antibodies to neurofilament (NF) triplet proteins~,t0-~2,1s,lsj0, uhiquitin4,0.ts.23.aT,~0 tubulin and high molecular weight microtubule-associated proteins (e.g. MAPs la and 2)4.t~,2~, but not with antibodies to tau 's'12.lS'z3'zTJ°.Although the uitrastructure of LBs is dominated by aggregated filaments that appear to be derived, at least in part, from NF proteins, LBs have yet to be isolated, purified and subjected to biochemical analysis. Hence, it has been necessary to employ immunohistochemical methods to dissect the composition of LBs. The idea that Alzheimer's disease (AD) and PD may overlap has been debated for a number of years, It
has been argued that AD and PD may share some common pathogenic mechanisms, or that the presence of one disease may predispose an individual to acquire the second4,'j,lq,2~','~°,~s,and even that AD and diffuse Lewy body disease (DLBD) belong to the same spec. trum of disease processes~,1~,Some studies suggest that concomitant AD and PD pathologies occur with a frequency greater than chance alone predicts s,7,t6`2°,2o, while others have suggested that these co-occurrences are statistically non.significant t4, Many, but not all, PD subjects presenting with clinical signs of dementia exhibit cortical pathology typical of AD ts't4't~,3s, and subcortical as well as cortical LBs also occur in a percentage of AD patients 6,~,t4,t~,:~,2s, While the co-existence of AD and PD pathologies in the same patients is not uncommon, the significance of this association is not well understood, Only recently has it been shown that senile plaques (SPs) in the hippocampus of a subset of demented PD subjects exhibit the same molecular features as those found in SPs of the AD hippocampus -~, rather than the features found in the SPs of neurologi-
* Present address: Department of Neurology, Motojima General Hospital, eta, Gunma 373, Japan. ** Present address: Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912-2000, USA. *** Present address: The Upjohn Company, Kalamazoo, MI 49001, USA. Correspondence: J.Q. Trojanowski, Department of Pathology and Laboratory Medicine, Division of Anatomic Pathology, The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
387 TABLE I
;;'ammary of antibody probes This table summarizes the immunochemical and immunohistochemical properties of the antibodies used in this study, aa, amino acid; BAP, beta-amyloid peptide; BAPP, beta-amyloid precursor protien; COOH, carboxT; LBs, Lewy bodies; mAbs, monoclonai antibodies; NFTs, neurofibrillary tangles; SPs, senile plaques.
Antibody
Specificity
LN21, 27, 39
These 3 mAbs are to epitopes within the first 100aa of BAPPs 695, 751 and 770; they label neurons, glia, extracellular NFTs, LBs, and the corona of SPs Antisera to aa 18-35 of BAPP 695; it labels the corona of SPs Antisera to aa 301-319 of BAPP 695 (glycosylation site); it labels the corona of SPs Antisera to va 1-42 of BAP; it labels NFTs, diffuse plaques and SPs mAb to aa 1-28 of BAP; it labels NFTs, diffuse plaques and SPs mAb to NF-M phosphorylation dependent COOH tail domain; it labels LBs but not NFTs Anti-ubiquitin mAb; it labels LBs, NFTs, neuropil threads, and SPs
Up84
ups6 UpI07 Amy33 RMO32 MABI510
cally normal controls. Further, the cortical LBs found in some AD patients contain the same constellation of NF protein epitopes that are found in cortical and SN LBs in PD patients Is'3°. This suggests that both SPs and LBs in AD and PD may result from linked pathological events. To pursue this issue further we probed cortical ~nd SN tissue samples from PD, AD/PD, and DLBD patients, as well as from neurologically normal control cases using antibodies to the beta-amyloid peptide (BAP) and other BAP precursor (BAPP) epitopes to determine if the BAP or its precursors were contained in the LBs found in these tissues. Monoclonal antibodies (mAbs) to the recombinant form of the full-length 695 a~i:lo acid (aa) BAPP were characterized and described elsewhere ~'a. These included 3 well.characterized mAbs (LN21, LN27 and LN39) that recognize epitopes within the first 100 aa of
',
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,.
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the amino-terminus of all three major BAPPs (BAPP695, BAPP751, and BAPP770). None of these mAbs cross-react with *;,,*ergent-insoluble cytoskeletal proteins derived from h,,nan brain. Additional antibodies were used that were raised to synthetic peptide homologs of different BAPP domains. These included: rabbit antisera Up84 (raised to aa 18-35 of BAPP695), Up86 (raised to aa 301-319 of BAPP695), and Upl07 (raised to aa 1-42 of BAP) ~. A second probe for BAP was also used, the mAb AMY33 (raised to aa 1-28 BAP) 3a. Another mAb, RMO32, which recognizes a phosphate-dependent epitope in the tail region of the middle molecular weight NF protein (NF-M) ms's°, was used since it specifically detects LBs and does not recognize neurofibrillary tangles (NFTs). Finally, a commercially available anti-ubiquitin mAb (Chemicon MAB 1510) which labels NFTs and LBs was employed
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Fig. 1. a: two LBs in the substantia nigra pars compacta are labeled by LN21. Note that the peripheries of the LBs are intensely stained (indicated by arrows), b: a cortical LB (parahippocampal gyrus) is shown here labeled with LN21 (arrow head)• In contrast to substantia nigra LBs those in the cortex are homogeneously labeled by the LN mAbs; note the uniform staining of the LB. c: the two LBs shown here (arrows) are unlabeled by UP107. The sections in these photomicrographs were counterstained with hematoxylin. Scale bars ffi 10 ~m.
388 as well is'3°''~. The properties of these antibodies are summarized in Table I. Brains were collected at autopsy from 14 PD cases (i.e. 4 non-demented PD patients, 5 demented PD patients without AD pathology, and 5 demented PD patients with AD pathology), 3 A D / P D cases (i.e. demented patients with a clinical diagnosis of AD and postmortem findings of AD and PD pathology), and 10 control patients without AD, PD or other neurodegenerative diseases. One case of DLBD was also studied. The clinical data and quantitative epitope analysis of hippocampal SPs in these patients has been described in detail elsewhere 3. The brains were either fixed in 10% neutral-buffered formalin, Bouin's solution, 70% ethanol/150 mM NaCI, or denatured by microwave energy. Both the fixation protocols and the performance of the antibodies used here with tissues denatured in these fixatives have been previously described ~-3.~s.3°.32'33. The tissue blocks were embedded in paraffin, sectioned at 6 ~tm, and then probed with the different antibodies using nearly consecutive sections according to previously published immunoperoxidase procedures ~-3'18'24'25'3°'32'33.Some sections were pretreated with 90% formic acid for 5 min prior to immunostaining to improve detection of BAP 2'32. The diagnosis of AD was established using the criteria recommended by the National Institute on Aging 2t, while PD and DLBD were diagnosed as described earlier*.tH.30. As reported previously 1°3, the anti-amino.terminal BAPP mAbs LN21. LN27, and LN39 stained the corona of SPs, neuronal cell bodies, neuronal processes, gila) and extracellular NFTs. Additionally, all three mAbs stained both SN and cortical LBs. Notably, in 'typical' SN LBs, the periphery of the LB core was intensely labeled, while the central core and halo of these LBs appeared unstained at the light microscopic level (Fig.
la). The BAPP immunoreactivity was present within SN LBs of all 14 PD patients, the 3 AD cases that had concomitant PD pathology in the SN, and the DLBD case. RMO32 and the anti-ubiquitin mAb labeled these LBs in adjacent sections. Corti¢ai LBs exhibited homogeneous or slightly granular staining with LN21, LN27 and LN39 (Fig. lb). immunolabeled cortical LBs were found in all 14 PD patients, the 3 AD patients with PD neuropathology, and the DLBD patient. Similar staining was not seen in the brains of the 10 control cases, all of which lacked SN and cortical LBs (as defined by H& E, RMO32, and anti-ubiquitin staining). Cortical and SN LBs were negative when probed with the anti-BAP Abs Upl07 and AMY33, even after pretreatment of the sections with formic acid. in contrast,
diffuse amyloid plaques, SPs and NFTs were stained with Upl07 and AMY33 on these same sections. This suggests that the amyloidogenic BAP does not accumulate within LBs (Fig. lc). The cortical LBs were distinguishable from tangleladen neurons, dystrophic processes in the neuropil and SPs using RMO32 3'~s'3°. Furthermore, only a subset of LBs appear to contain BAPP epitopes, since fewer LBs were labeled by LN21, LN27 and LN39 than with RMO32 and the anti-ubiquitin mAb. Relative to RMO32, approximately 30-40% of SN LBs and 1020% of cortical LBs were detected with these BAPP mAbs. However, this is consistent with the immunodetection of other proteins in LBs, including many, if not most, NF protein epitopes ~s'a°. This phenomenon may reflect post-translational modifications of NF and BAPP proteins affecting their tertiary conformation and epitope antigenicity. Additionally, LBs which lack NF and BAPP immunoreactivity may contain the epitopes for which they are being probed, but they may be below the threshold level required for detection with these specific antibodies. Alternatively, the association of BAPP with LBs may occur secondary to LB formation. In contrast to the LN series of mAbs, no convincing immunostaining was obtained with either Up84 or Up86. However, it has been noted previously that these polyclonal antibodies are less sensitive than the LN mAbs in recognizing BAPPs 1'3. Thus, relatively low levels of BAPPs in LBs may go undetected with these antibodies. This is the first study to describe the presence of BAPPs in cortical and SN LBs. However, it is not clear if our data imply that amino-terminal fragments or full-length BAPPs are present in these LBs. If BAPPs play a role in the formation of LBs, it could be a result of the ability of BAPPs to bind to and interact with cytoskeletal proteins. This possibiliW is raised by the observation that BAPPs partition with detergent-insoluble cytoskeletal preparations 2~. Whether BAPPs interact directly with cytoskeletal elements or through other proteins is not clear, it is also possible that the findings reported here represent the interaction of BAPP-containing vesicles with the cytoskeleton. Since Refolo et al. 2~ have provided evidence that BAPP/cytoskeleton interactions are microtubule-dependent, and the full-length BAPPs are integral membrane proteins, it is possible that BAPPs reside in cytoplasmic vesicles as integral membrane proteins that interact with the cytoskeleton. Duffy and Tennyson s have shown that large numbers of small vesicles are localized to the periphery of the central core of SN LBs. Thus the distribution of BAPP immunoreactivity in SN LBs seen in this study may reflect the accumulation of BAPP
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containing-vesicles within LBs. If LBs are in part formed through the disturbance or collapse of the neuronal cytoskeleton, then vesicles with membranes rich in BAPP may be associated with one or more of the cytoskeletal components that form the filamentous meshwork of the LB. That a cytoskeletal disturbance could be initiated by one or more of the BAPPs or the amyloidogenic BAP itself is suggested by studies of BAPs injected into the brains of rats which showed alterations in tau protein immunoreactivity 22. Further, BAP immunoreactivity is frequently associated with N F T s I-3. Therefore, disturbances in BAPP expression or metabolism may trigger pathogenic event~ involving the neuronal cytoskeleton. Recently, the amyloidogenic protein found in familial amyloidosis, Finnish-type (FAF), has been partially sequenced and found to be identical to a portion of gelsolin except for a single aa substitution 34. An antiserum raised to the purified FAF amyloid was found to label SN and cortical LBs in PD and DLBD. The finding of gelsolin, an actin binding and regulatory protein, or a gelsolin-related molecule in LBs is additional evidence that the LB may represent a general disturbance or collapse of the neuronal cytoskeleton. Further, the identification here of BAPPs in LBs, together with the data on FAF, show that two distinctly different amyloidogenic peptides accumulate in LBs. This constitutes additional evidence linking disruption of the neuronal cytoskeleton and amyloidogenesis in neurodegenerative diseases. The authors wish to thank Mr. G, DiDario l'~r help with the figures and Ors, G. GottUeb, M. Grossman, R.C. Gur, !1.1. Hurtig, as well as Ms, M, Phoenix R,N., and the residents and staff of the Division of Anatomic Pathology who aided in the collection of postmortem tissues, We wish to add special thanks to the patients and their families who made our work possible through their generous efforts to foster research, and the Philadelphia Alzheimer's Disease and Related Disorder Association Inc. This work was supported by Nll+I Grants AG-09215 (JOT), AGI0124 (JOT) and AG.05465 (WDH). 1 Arai, H., Lee, V.M.-Y., Messinger, M.L., Greenberg, B.D., Lowery, D.E. and Trojanowski, J.O., Expression patterns of betaamyloid protein (beta-APP) in neural and non-neural human tissues from Alzheimer' s disease and control subjects, Ann. Neurol., 30 (1991) 686-693. 2 Arai, H., Lee, V.M.-Y., Otvos Jr., L., Greenberg, B.D., Lowe~, D.E., Sharma, S.K., Schmid'~ M.L. and Trojanowski, J.O., Defined neurofilament, tau and beta.amyloid protein epitopes distinguish Alzheimer from non..Alzheimer senile plaques, Prec. Natl. Acad. Sci. USA, 87 (1990) 2249-2253. 3 Arai, H., Schmidt, M.L., Lee, V.M.-Y., Hurtig, H.I., Greenberg, B.D., Adler, C. and Trojanowski, J.Q., Epitope analysis of senile plaque components in the hippocampus of patients with Parkinson's disease, Neurology, in press. 4 Bancher, C., Lassmann, H., Budka, t!., Jellinger, K., Grundkelqbal, I., lqbal, K., Wiche, G., Seitelberger, F. and Wisniewski, H.M., An antigenic profile of Lewy bodies: immunocytochemical indication for protein phosphorylation and ubiquitination, J. Neuropathol. Exp. Neurol., 48 (1989) 81-93. 5 Boiler, F., Mizutani, T., Roessmann, U. and Gambetti, P.,
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