Epitope Map of Neurofilament Protein - Europe PMC

American Journal of Pathology, Vol 1-39, No. 1, July 1991 Copyright © American Association of Pathologists

Epitope Map of Neurofilament Protein Domains in Cortical and Peripheral Nervous System Lewy Bodies M. L. Schmidt,* J. Murray,t V. M-Y Lee,* W. D. HiII,* A. Wertkin,* and J. Q. Trojanowski* From the Division of Anatomic Pathology, Department of Pathology and Laboratory Medicine,' and the Department of Anatomy, t The University of Pennsylvania School of

Medicine, Philadelphia, Pennsylvania

A subset of demented elderly patients exhibit large numbers of cortical intraneuronal inclusions similar to the neurofilament (NE)-rich Leuy bodies (LB) found in pigmented subcortical neurons of patients with Parkinson's disease (PD). Because these cortical inclusions may contribute to the emergence of cognitive impairments in afflicted individuals, the authors mapped the distribution of NF epitopes in these so-called cortical LBs. This was done using ethanolfixed tissues and a large library of monoclonal antibodies (MAbs) with well-characterized binding specificities to various regions of each NF triplet protein Cortical LBs were examined by light, confocal, and electron microscopy, and they were compared with the subcortical LBs of PD and LBs in the peripheral nervous system (PNS). Monoclonal antibodies specific for the rod regions of each of the three NE subunits, or for phosphate-dependent and independent antigenic sites in the tail region of the high(NE-H) and middle- (NF-M) molecular weight (Mr) NF subunits as well as other MAbs to the extreme COOH terminus of NF-L and NE-M or the head region of NE-M labeled a variable number of cortical LBs. Remarkably one of these anti-NF MAbs, RM032, which recognized a phosphorylated epitope in the tail region of NF-M, immunolabeled nearly all cortical LBs, whereas each of the other anti-NE MAbs never labeled more than 10% of ubiquitin- or RM032-positive cortical LBs. Further LBs in the PNS resembled those in the central nervous system (CNS) in their immunologic properties, and LBs in both sites were dominated by filamentous aggregates at the ultrastructural level. These findings suggest that NE proteins are profoundly altered during their incorporation into cortical and PNS LBs. Further the

authors here identified immunologic and ultrastructural properties common to cortical LBs, PNS LBs, and classic substantia nigra LBs in PD. The accumulation of filamentous, perikaryal inclusions rich in NE proteins at diverse sites in the CNS and PNS of patients with a variety of neurodegenerative disorders suggests a widespread disruption of NF metabolism or transport. (Am J Pathol 1991, 139:53-65)

In recent years, a subset of demented elderly patients were shown to exhibit widespread Lewy bodies (LB) or Lewy body-like inclusions in neurons of the cerebral cortex, and it has been speculated that the accumulation of these inclusions in cortical neurons may lead to cognitive impairment in a large percentage of the elderly with dementia.1 7 Ikeda et al,8 however, reported a case of Parkinson's disease (PD) without intellectual or behavioral deterioration in which numerous LBs were found in several cortical areas. Nevertheless neurodegenerative changes characteristic of Alzheimer's disease (AD) or PD have been reported to be associated with a variable number of cortical LBs in some patients. Thus a somewhat diverse and incompletely characterized spectrum of dementing diseases associated with cortical LBs has emerged, and cortical LBs have received increased attention because they may contribute to cognitive impairments.49 Indeed Perry et a15 estimate that senile dementia of the LB type may constitute as much as 20% of the institutionalized demented population older than 70 years. Despite the recent recognition of these disorders, AD with few or no cortical LBs still remains the most common form of dementia in the elderly. Part of the reason for uncertainties about the contribution of cortical LBs to dementia can be attributed to the fact that little is known about the composition and structure of cortical LBs. Indeed cortical LBs were difficult to Supported by NIH grants AG-09215 MH-43880. Accepted for publication February 21, 1991. Address reprint requests to John Q. Trojanowski, MD, PhD, Hospital of the University of Pennsylvania, Division of Anatomic Pathology, Maloney Building Basement, Room A009, 3400 Spruce St., Philadelphia, PA 19104-4283.

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54 Schmidt et al AJPJuly 1991, Vol. 139, No. 1

identify in the past because they stained poorly or not at all with eosin. In contrast, brainstem LBs are intensely eosinophilic, although different types of eosin staining methods disclose varying numbers of cortical LBs.5 Recently the identification of cortical LBs was facilitated by the use of monoclonal antibodies (MAbs) or antisera to ubiquitin1 and to phosphorylated neurofilament (NF) proteins.2 These antibodies visualized cortical LBs, and the ultrastructural appearance of these LBs was similar to that of subcortical LBs.2 8'1>12 Notably there is regional variation in the density of cortical LBs, and cingulate cortex contains the highest number, followed by insular, temporal, and frontal cortices.2 5'7'8'11'13 Like LBs in the substantia nigra and the locus coeruleus, cortical LBs occur primarily in tyrosine hydroxylase-synthesizing neurons, which presumably are catecholaminergic.14 Lewy bodies also occur in neurons of the nucleus basalis of Meynert, however, which is known to contain cholinergic and other non-monoaminergic neurons.7' 15 Although LBs can develop in central nervous system (CNS) neurons with different neurotransmitter phenotypes, they also have been found outside the CNS, ie, in peripheral nervous system (PNS) neurons. This has clinical significance because these PNS neurons are readily accessible to biopsy, which could lead to the development of diagnostic strategies for a more precise antemortem recognition of LB dementia. Therefore we investigated LBs in both the PNS and cortex to establish whether they are similar to the classic LBs of PD and to determine if they occur in other neuro-

degenerative conditions. Because classic LBs are composed primarily of filamentous aggregates rich in NF proteins, we analyzed the NF epitopes contained in cortical and PNS LBs using immunohistochemistry in conjunction with light and confocal microscopy. Finally we also studied their composition at the ultrastructural level. A comparison of these findings with the results of a similar investigation of LBs in the substantia nigra of PD patients16 showed many similarities and some differences between the latter LBs and those found in cortex and the PNS.

Material and Methods Tissue Collection and Preparation Cortices from 56 patients with and without neurodegenerative diseases were collected at autopsy. Simultaneously tissue samples of various peripheral organs and sensory ganglia were obtained. The diagnosis, number of patients in each disease category, age, sex, postmortem interval, fixative, and tissue samples studied are listed in Table 1 for the CNS LBs and in Table 2 for the PNS LBs. The criteria used to assign patients to different neurodegenerative diseases categories have been described elsewhere.17"20 Briefly, the diagnosis of AD was based on the National Institute on Aging consensus criteria as previously reported.21 The diagnosis of PD and specific Parkinson plus syndromes (ie, progressive supranuclear palsy) was based on the presence of parkin-

Table 1. Patient Information for Cortical LB Studies

Diagnosis AD

No. of cases in each group 29

PD/AD

6

PD*

7

DLBDt PSPt

5

Down/AD

4

Control

6

Sex

Age range and average in years

17 F 12 M 2F 4M 3F 4M 1 F 2F 3M 1 F 3M 2F 4M

65-92 av = 78 65-75 av = 70 53-85 av = 71 58 69-78 av = 75 54-78 av = 66 71-92 av = 81

Fixatives/no. of hemispheres studied

PMI range and average in hours

B

E

M

2-23

15

12

6

LBs 7

3

3

0

6

5

3

1

7

0 3

1 2

0

1

1

0

3

1

1

0

4

2

0

av = 11 4-22 av = 10 3.5-17 av = 9 10.5

2.5-15 av = 7 7.5-15.5 av =12 10-23.5 av = 15

No. cases with cortical

* Three PD patients were demented but had no AD pathology. t This patient had diffuse Lewy body disease. t One PSP patient also had AD pathology and another one was demented without AD pathology. § No LBs were detected in controls using the anti-ubiquitin MAb; 1 LB was found using MAb RMO1 11. One or both hemispheres from the patients listed here were screened with an MAb to ubiquitin to identify cases with cortical LBs for subsequent epitope mapping using anti-NF MAbs. Temporal and midfrontal cortices were evaluated in each hemisphere. Furthermore sampling from 45 orbital frontal, 32 entorthinal, 17 parietal, 15 motor, 14 occipital, and 3 cingulate cortices were evaluated for the presence or absence of LBs. AD, Alzheimer's disease; PD/AD, Parkinson's disease (PD) combined with Alzheimer's disease or vice versa; B, Bouin's fixative; DEM, dementia; DLBD, diffuse Lewy body disease; DLDM, dementia lacking distinct morphology; DOWN/AD, Down's syndrome combined with Alzheimer's disease; E, 70% ethyl alcohol with 150 mmol/l NaCI; M, denaturation by microwaves; No., number; PMI, postmortem interval; PSP, progressive supranuclear palsy.

NF Epitopes in CNS and PNS Lewy Bodies

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Table 2. Patient Information for PNS LB Studies Age range No. of and average cases Sex in years Diagnosis AD

3

PD/AD

6

PD*

4

DLBD PSP

1 2

Down/AD

4

DLDM Control

1 3

2F 1M 2F 4M 2F 2M 1M 1F 1M 3F 1M 1M 2F 1M

67-78 av = 72 65-75 av = 70.2 65-77 av = 70.5 58 65-77 av = 71 53-58 av = 56.5 69 71-91 av = 79.3

PMI range and average in hours

14-20 = 16.8 4.5-21 av = 10.3 5-17 av = 9.3 10.5 8-9 av = 8.5 6-13 av = 9.1 20 11.5-13 av = 12

No. of

Fixative

cases with

B

E

M

PNS LBs

0

4

0

0

3

3

0

3

av

0

1 0

1

0

1

2

0

0

0

0

3

1

0

0

1

0

0

1

3

0

0

* Three PD patients were demented but had no AD pathology. This table summarizes the information on these patients from whom peripheral tissues were obtained to probe for PNS LBs. Abbreviations are shown in the legend to Table 1.

sonism, other clinical findings, and the histopathology.22 The diagnosis of diffuse LB disease and dementia lacking distinctive histologic features was based on criteria described in several recent reports.4'5'23'24 Tissue blocks of various neocortical areas, in addition to hippocampus, were excised and placed either in Bouin's solution or 70% ethyl alcohol (ETOH) containing 150 mmol/l (millimolar) sodium chloride (ETOH/NaCI). In some cases, tissue blocks were placed in 0.1 mol/l (molar) TRIS-HCI buffer pH 7.6 containing 150 mmol/l NaCI (TBS), denatured by microwave irradiation as described,25 and subsequently placed in 70% ETOH. Within the following 3 to 4 hours, all of the tissue blocks, except those denatured by microwave irradiation, were cut to a thickness of 3 to 4 mm, fixed for a total of 16 to 20 hours, and then transferred to a Shandon Hypercenter to infiltrate them with paraffin according to a previously described 18-hour embedding protocol.25 Microwave denaturation was carried out with the tissues immersed in 0.1 mol/l TRIS-HCI buffer, pH 7.6. Then these tissues were immediately transferred to the Shandon Hypercenter for infiltration with paraffin.25

RMO1 26. The binding of the primary antibodies was visualized either with a secondary antibody labeled with rhodamine isothiocyanate or by using the peroxidaseantiperoxidase procedure (PAP) and diaminobenzidine (DAB) or 4-chloro-1-naphtol (4CIN) as described.17'26 Medium prepared using the latter chromagen contained 0.05% 4CIN added to 1 part methanol and 5 parts 0.1 mol/l TRIS-HCI buffer pH 7.6 containing 0.1% hydrogen peroxide. Procedures to enhance staining with some MAbs included the treatment of tissue sections with 0.005% trypsin (Worthington, Freehold, NJ) in TBS for 30 minutes at 37 C27 before incubation with the primary antibody. Tissue sections containing LBs visualized with DAB were cover slipped in Permount (Fisher Scientific, New York, NY), whereas tissue sections containing LBs demonstrated with 4CIN or rhodamine-labeled second antibodies were cover slipped in Aqua-Mount (Lerner Laboratories, Pittsburgh, PA). The sections stained for immunofluorescence were analyzed with a Bio-Rad confocal imaging system. The PAP-treated tissue sections were lightly counterstained with hematoxylin. The size of cortical LBs was measured with the aid of an ocular micrometer at a magnification of x400.

Light and Confocal Microscopy Electron Microscopy Tissues were cut with the microtome set for a nominal thickness of 6 ,u. Tissue sections were incubated overnight at 4°C with a given MAb, or in some cases, with polyclonal antisera. To determine whether different MAbs stained distinctly, as contrasted with overlapping populations of LBs, some sections of cingulate cortex were incubated with a 1:1 mixture of a anti-NF MAb that almost exclusively stained LBs (RM032) and one of the following other anti-NF MAbs: RMO60, RMO90, RMO1 11, and

Twenty intraneuronal NF-positive cortical LBs as well

as

two NF-positive PNS LBs were selected for examination at the electron microscope (EM) level. To do this, the inclusions were first identified at the light microscopic

level by immunocytochemistry using either DAB or 4CIN, and then photographed using brightfield or Hoffman optics, deparaffinized, and subsequently re-embedded in EMbed 812 (Electron Microscopy Sciences, Fort Wash-

56 Schmidt et al AJPJuly 1991, Vol. 139, No. 1

ington, PA). Sections containing LBs visualized with

4CIN, were immersed in 100% ETOH on a shaker overnight to dissolve the chromagen before embedding in EMbed 812. All sections used for EM were dehydrated in an alcohol series, transferred to propylene oxide, and placed in increasing concentrations of EMbed 812. Finally, a gelatin capsule filled with EMbed 812 was placed over the region of the section containing the inclusions of interest and the resin was polymerized at 600C overnight. Sections were cut at approximately 100 nm and stained with 8% aqueous uranyl acetate and 0.5% lead nitrate. The thin sections were viewed in a Hitachi H-600 electron microscope at 75 kV.

Antibody Probes To conduct this epitope analysis, we used 324 MAbs to phosphorylated and nonphosphorylated NF epitopes, 2 MAbs to tau protein (T), and one MAb to the high molecular weight microtubule associated protein 2 (MAP2). These MAbs were generated over the last 10 years by V. M.-Y. Lee and coworkers. Notably the location and phosphorylation state of the epitopes recognized by these MAbs as well as, other properties of these MAbs (eg, whether or not they cross react with unrelated antigens) have been described in a series of previous publications. 17,18,22,25,28 36 A polyclonal antibody to the COOH terminal 20 amino acids in NF-L (designated RAS) was produced in rabbit and column purified.35 Additional antibodies from other sources also were used here and they included: Alz5O, a MAb initially used to identify A68 proteins,?037'38 which now is known to be a derivatized form of tau37 (a gift of Dr. P. Davies), and an MAb to ubiquitin produced by Shaw and Chau39 (Chemicon International, Inc, Temecule, CA). Finally, we also used a polyclonal antibody to the a melanocytestimulating hormone (otMSH), which we purchased from Instar Corp., Stillwater, MN, because it has been shown to cross-react with the first four amino acids at the amino terminus of NF-M.40 Most of the MAbs were used as undiluted supernatant, but other antibodies were used at one of several dilutions, ie, Alz5O and RAS 1:50, aMSH 1:1000, and anti-ubiquitin 1:80000. Remarkably one anti-NF MAb, RM032, labeled LBs almost to the exclusion of other normal and abnormal structures (see Results for details). This MAb was originally raised to rat NF-M immunogens and was shown to be specific for a phosphate-dependent epitope in the tail domain of NF-M.32 Because of its ability to preferentially label human LBs, the specificity of RM032 was probed in greater detail here. First RM032 was shown to recognize human NF-M in Western blots of enriched NF preparations obtained from human spinal cords (Figure 1 A). Notably RM032 recognized a phosphate-dependent

A.v -+

r.r

BI0

.

....

. .,dj

.I Figure 1. A and B depict Western blots ofNFpurifiedfrom human spinal cord The NF preparationi in lanes 2 of both panels were treated uith alkaline phosphatase before electrophoresis. The blots uere probed uith JlAb RIlO-32 A and RA10308 (B). RM0308, u'hicb binds- to ani epitope uithin the multiphosphorylation repeat domain of NPAl,f uwas u-sed as positive control. C depicts an immunoblot of homogenized humani cingulate cortex (lane 1) and human occipital cortex (lane 2) reacted uith AMAb RM032. D shou's a Coomassie blue-stainied 7.5% polvacrylamide gel containing homogenized antd electrophoresed human cingulate (lane 1) and buman occipital cortex (lante 2). Protein (0.8 mg) uwas loaded in each lane. 7The arrowu in A marks the 1 70-kd banid ofhuman NF-M.

epitope in human NF-M just like other similar MAbs in our antibody library (compare Figure 1 A and B). This was demonstrated by the marked diminution of the NF-M immunoband produced by RM032 after dephosphorylation of the NF-enriched fraction with alkaline phosphatase, as shown in Figure 1. Efforts to localize further the RM032 epitope in human NF-M were conducted by probing Western blots of cyanogen bromide and chymotryptic digests of purified human NF-M. Surprisingly RM032 did not recognize any NF-M cleavage products (data not shown). RM032 did not show any evidence of cross-reactions with other human brain proteins, however, the only immunobands labeled by this MAb in whole homogenates of human cortex (eg, cingulate and occipital cortex, as shown in Figure 1 C) corresponded to NF-M only. Thus our data indicate that RM032 binds to a phosphate-dependent epitope in human NF-M. Although the precise topographic location of the RM032 epitope in NF-M remains to be identified, the findings presented here suggest that it is located close to the junction of the core and tail domains of NF-M.

Results

Light Microscopy Cortical LBs immunostained with the anti-NF, antiubiquitin, and anti-MAP2 antibodies appeared as homo-


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AJPJuly 1991, Vol. 139, No. 1

geneously or granular staining intraperikaryal round or elliptical structures. In most cortical neurons that contained LBs, the nucleus was crescent shaped and displaced to the cell margin (Figure 2). A large portion of cortical LBs that stained with the anti-NF MAbs showed an immunostained core surrounded by an unstained halo, whereas only an occasional cortical LB was homogeneously NF positive. In both types of cortical LBs, lighter stained central areas were sometimes observed (Figure 2A). Monoclonal antibodies to ubiquitin, MAP2, and a unique epitope in NF-M (ie, RM032) immunostained cortical LBs uniformly, ie, a halo was not discernable. Some of these cortical LBs, however, exhibited a less intensely stained central region. Measurement of the cortical LBs with and without halos showed similar external diameters, and the cores of those LBs with halos were consistently smaller than LBs without halos (Table 3). The anti-ubiquitin MAb and RM032 also stained highly irregularly shaped (Figure 2C, D) cortical LBs. Most LBs were found in layers IV to VI in most of the cortical areas studied. They were rarely seen in layer Ill and never in layers and 11. Cingulate cortex was exceptional in this regard,

as LBs also occurred in more superficial layers. Most neurons containing LBs lacked apical dendrites, although a few of these neurons (especially those at the junction of cortical layers Ill and IV) had more typical pyramidal features. The number of cortical LBs varied from case to case and in different cortical regions. For example, more than 100 RM032-positive LBs per tissue section (ie, within a strip of cortex approximately 30 mm2 in area) were found in the cingulate cortex of one AD/PD patient and frontal cortex of the patient with diffuse LB disease. Furthermore in adjacent sections from these cortices, anti-NF MAbs other than RM032 labeled only up to about 20 LBs with halos. When adjacent sections of these same cortical regions were probed with RM032 mixed with equal amounts of another anti-NF MAb (ie, RMO60, RMO90, RMO1 11, or RMO1 26 as described above), homogeneously stained round or irregularly shaped LBs prevailed, and LBs with a dark core surrounded by an unstained halo were not observed. This indicates that RM032 immunostains the same population of cortical LBs as the other anti-NF MAbs, in addition to a much larger population of LBs that are not stained by the other anti-NF MAbs. Peripheral nervous system LBs were examined less extensively because of their rarity, but they all were homogeneously immunostained with anti-NF MAbs RM032, RMO1 00, or the MAb to ubiquitin. In contrast to cortical LBs, which were found exclusively in neuronal perikarya, a number of PNS LBs were located in neurites (data not shown).

Confocal Microscopy '.'C.

D

,s: , ' . .

a.'.

W.i

Figure 2. Cortical LBs with a core and halo are illustrated in A and B and irregularly shaped inclusions in C and D. In all LBcontaining neurons shown, the nucleus is displaced to the cell margin. A, C, D: Cingulate cortex of an 80-year-old patient with AD and PD. B: Temporal cortex of a 66-year-old AD patient. A: MAb RM066 binds to epitopes in the LB core, in axons, and in the neuropil but not in the cytoplasm. B: MAb RM0126 labels the LB core, the cytoplasm of the neuron, and the neuropil. A somewhat lighter center is visible within the core of this LB. C, D: Two examples of delto inclusions immunostained by MAb RM032. (A round to ellipsoidal cortical LB labeled by this MAb is illustrated in Figure 4.)None of these inclusion bodies have halos. Neither the cytoplasm nor neuropil or neurites are stained by this MAb. Space bar = 5 [t.

Confocal microscopy was performed on cortical tissues labeled by immunofluorescence. Photographs taken at O.5-p depth intervals confirmed the spherical to ellipsoidal shapes of cortical LBs. Monoclonal antibodies to ubiquitin and RM032 labeled cortical LBs homogeneously throughout their depth, although more and less intensely labeled central areas were observed. In cortical LBs immunolabeled with MAbs to NFs, confocal microscopy demonstrated intensely immunoreactive cores that were completely surrounded by nearly unstained halos. Some of these cores contained less intensely stained areas of various shapes (Figure 3).

Electron Microscopy Thin sections showed that round and irregularly shaped cortical LBs were composed of amorphous material and 10- to 20-nm filaments with irregular contours. Aggre-

58

Schmidt et al

AJPJuly 1991, Vol 139, No. 1

Table 3. Size of Cortical LBs Immunostained with Monoclonal Antibodies (MAb) to Ubiquitin andNFs Average Average diameter of diamater in , core in ,. MAb Anti-ubiquitin RM032 RMO60

8.07 9.40 8.80

6.32

Fixative

Bouin's solution ETOH/NaCI ETOH/NaCI

The external diameter of the entire LB and the LB core of 15 LBs each stained with the MAbs listed in this table were measured and the average values are shown here. Note that the size of the entire LBs immunostained with the anti-ubiquitin or RM032 MAb was always larger than LB cores stained by RMO60. Abbreviations are the same as in Table 1.

gates of amorphous material were found in the centers of some cortical LBs, whereas the periphery frequently consisted of a loose meshwork of amorphous material in a filamentous or strandlike formation. An intermediate zone contained many filaments and closely associated amorphous material. The transition between each of the zones described above was gradual. Representative light and electron microscopic photographs of a cortical LB are shown in Figure 4. More electron-dense material occurred in cortical LBs originally treated with DAB, and this corresponded to deposits of DAB reaction product. Electron microscopy was performed on one LB found in Auerbach's plexus of the colon. This LB was originally identified using 4CIN, and it contained almost exclusively filamentous components and irregular unstained central areas (Figure 5). A second PNS LB visualized with DAB also had a dense filamentous core surrounded by accumulations of electron-dense material, which probably represented DAB. The outer zone of this LB was composed of a loose meshwork of filamentous strands of amorphous material similar to those seen in cortical LBs.

NF Epitopes in Cortical and PNS LBs Initially the anti-ubiquitin MAb was used to determine which cortices contained LBs. Sections of frontal cortex of one AD patient with ubiquitin-positive LBs then were used to screen 324 antibodies to NF proteins to identify those that bind cortical LBs. Of the 324 MAbs tested, 77 bound LBs in cortex fixed with ETOH/NaCI and only 11 MAbs stained these structures in the equivalent cortical area from the opposite hemisphere fixed with Bouin's solution. For this reason we used only the ETOH/NaCI-fixed cortices to explore further the NF epitopes present in cortical LBs. In contrast, the anti-ubiquitin MAb yielded similar immunostaining of cortical LBs in both ETOH/NaCI and Bouin's fixed tissue. Of 77 MAbs that stained cortical LBs, we selected 45 for further study based on their ability to detect well-defined, spatially separate epitopes in each NF triplet protein. The NF epitopes encountered in cortical LBs are listed in Figure 6. Briefly, epitopes that reside in the rod region of NF-L, NF-M, and NF-H, the tail region of NF-M and

NF-H, the amino terminus of NF-M, as well as the extreme carboxy terminus of NF-M and NF-L, were found in cortical LBs. Monoclonal antibodies to both the phosphorylated and nonphosphorylated state of the multiphosphorylation repeat motifs in NF-M and NF-H labeled cortical LBs. Three MAbs to epitopes spanning T protein (Alz5O, Ti 4, T46) did not stain cortical LBs (but see Galloway et aI41); however an anti-MAP2 (Ml 2) MAb did stain them. Peripheral nervous system LBs were infrequent and were probed to a limited extent, but ubiquitin and NF-M epitopes (RM01 00, RM032) were present in PNS LBs. In contrast to cortical LBs, however, RM032 (the MAb to an epitope at the junction of the rod and tail domain of NF-M) did not bind to as many PNS LBs as the MAb to ubiquitin (see below).

Cortical LBs in Subjects With and Without a Neurodegenerative Disease Cortical LBs were present in almost 25% of AD patients and in 100% of the PD patients examined in this study. Notably some of these PD cases exhibited sufficient concomitant numbers of senile plaques (SPs) or neurofibrillary tangles (NFTs) to warrant the combined diagnosis of PD and AD. As noted above, the number of cortical LBs was highly variable (ie, from only 1 in multiple different cortices to >200 in a 4- x 7-mm strip of cortex) from case to case and also among cortical areas of the same patient. The greatest variation in the number of cortical LBs was found in the PD patients. In most patients, including the AD group, cortical LBs occurred in the temporal as well as in the frontal lobe. Only moderate numbers of LBs occurred in the motor cortex. Lewy bodies were rare in the parietal cortex and extremely rare in the occipital cortex. Significantly only one of the six age-matched control patients was found to have any LBs. In this case, a single LB was in the temporal cortex. Cortical LBs were not found in any of the five progressive supranuclear palsy cases, nor in the three elderly Down's syndrome patients with AD. The number of immunostained cortical LBs also depended on the antibody used to demonstrate them. For example, when adjacent sections of midfrontal or cingu-


AlPJul4y

59

1991, Vol. 139, No. 1

Figure 3. Confocal microscopy images of tuwo cortical LBs immunolabeled with RMA032 (A) and RiO9O (B), respective/,i are depicted. a: Sixteen photographs taken at 0.5-pL intervals throughout the depth of an LB. The LB is homogeneousl/ stained by! RM032 and is spherical on one pole and ellipsoidal on the opposite pole. Autofluorescent lipofuscin granules are found in close proximit), to the LB. b: Eleven photographs taken at 0. 5-p intenrals of an LB labeled with RAM090. Only the core region of this LB is intetnselly immunostained and the halo is discernable in planes 3 to 9. A poorly defined less intensely stained area uwithin the core is l'isible in planes 5 to 9. Space bar = 5p.

60

Schmidt et al


demonstrated the highest numbers of cortical LBs. In contrast, the anti-ubiquitin MAb labeled an average of 52% (range, 33% to 73%) of these RM032-positive inclusions in adjacent sections. All of the other anti-NF MAbs stained a smaller percentage (0.7% to 7.5%, except for one case with 33%) of RM032-positive cortical LBs. Aside from cortical LBs, only rare neurites in gray and white matter and some dystrophic neurites in senile plaques were weakly stained by RM032. In contrast, the anti-ubiquitin MAb labeled cortical LBs as well as SPs, NFTs, and neuropil threads (NTs). Figure 7 demonstrates the numerous ubiquitin-positive SPs, NTs, and NFTs in hippocampus of one AD patient who had cortical LBs in several different neocortical areas. Notably SPs, NFTs, and NTs were not stained by RM032. Lewy bodies in PNS neurons occurred in six PD patients and in one patient with diffuse LB disease. Three of the PD patients had concomitant AD and the three other demented PD patients had no evidence of AD pathology. All LBs within the viscera were found in Auerbach's plexus. Adrenal LBs were restricted to the medulla. A single LB was encountered in one dorsal root ganglion, and none were seen in the trigeminal ganglia. The exact numbers of LBs observed per organ and disease state are summarized in Table 4.

Discussion

Figure 4. Light micrograph using Hoffman optics of a cortical LB immunostained witb MAb RMO 32 using 4ClN as chromagen. This section was not counterstained. Space bar = 25 pL. B: Lou-pouer electron micrograph of the LB depicted in A. The cell nucleus is in the upper right of the picture. Arrouwheads indicate lipofuscin granules. Space bar = 1 pL. C: Higher magnification of the area delineated in B demonstrating the amorphous and filamentous components of this LB. Space bar = 0.25 R.

late cortices from four patients (one AD, one PD, two PD with AD) were immunostained with RM032, the ubiquitin MAb, or one of eight different anti-NF MAbs, RM032

Although cortical LBs have been the focus of several recent studies,1 our study is the first to establish the extent to which each NF triplet protein is present in these inclusions, and we have significantly extended insights into the morphologic and molecular similarities between cortical, PNS, and brainstem LBs.1042'43 Furthermore we provide additional information on a variety of neurologic diseases in which they occur. For example, we found cortical LBs in all PD patients and in 25% of AD patients, but no cortical LBs were encountered in three elderly patients with Down's syndrome and AD or in five patients with progressive supranuclear palsy. In agreement with an earlier report, cortical LBs were found mostly in nonpyramidal neurons of the deeper cortical layers.42 The largest numbers of cortical LBs, however, were found in the cingulate cortex, and they were present in layers 11 through VI. Temporal and frontal lobe frequently contained cortical LBs, but they were less common in the postcentral gyrus and sparse in the other cortical areas studied here. Unlike subcortical LBs, cortical LBs stained poorly, if at all, with eosin,6 but antibodies to ubiquitin were highly effective probes for the demonstration of cortical, subcortical, and PNS LBs.1 464445 Ubiquitin binds to many different proteins targeted for adenosine triphosphate-dependent, nonlysosomal degradation, and ubiquitin immunoreactivity therefore is not a

NF Epitopes in CNS and PNS Lewy Bodies 61 AJPJuly 1991, Vol. 139, No. 1

Figure 5. The tissue was fixed in Bouin's solution, paraffin embedded, and immunostained with an MAb to ubiquitin using 4C1N as chromagen before preparation for EM. A: Light micrograph showing two PNS LBs. The arrouhead indicates the LB processed for EM micrograph. Space bar = 10 R. B: Low-power electron micrograph. This LB is composed mostly offilaments with central unstained areas. Space bar = 1 pL. C: High-pouer EM micrograph of the area delineated in B showing the mostly'filamentous structure of this LB. Space bar = 0.25p.

specific marker for LBs.4647 Indeed ubiquitin-positive globules were observed in patients with and without neurodegenerative diseases.48'49 Furthermore many different AD lesions (NFTs, SPs, NTs), which also occur in small quantities in many normal elderly individuals, were stained with anti-ubiquitin antibodies. Thus the unequivocal recognition of cortical LBs using anti-ubiquitin and light microscopy may be difficult. In contrast, our data suggest that the anti-NF-M MAb designated RM032, which intensely stained cortical LBs, but not NFTs, SPs, NA

RMS 12

NFL NH2

TA66 ROD

or NTs, may facilitate the unequivocal identification of cortical LBs (Figure 7). Cortical LBs immunostained with anti-NF antibodies (other than RM032) were round or oval and consisted of an NF-positive core surrounded by an unstained halo, and only rarely were they homogeneously stained. Both RM032 and the anti-ubiquitin MAb yielded homogeneously stained cortical LBs, some of which were round or oval, but most were irregularly shaped as originally described by Friederich Lewy.50 Round, oval, and irreg-

RAS

NA

COCH

RMO

44, 48,57 60,66,90,1 11, 128,1 40,1 46,1 50,1 54,184,186, 189 195,204,21 0,2 8,249,25 RMO RMO 46 1 00 TA54 aMSH 266,292,3 1 0 106,255 NFMINH2H ROD MPR T _ COOH _T OH

MPR

LRMO PHOS

NFM1+H

- RMO 39,40,224

32 123

TA 50

MPR

-

Figure 6. The structure of the low-, middle-, and high-molecular-weight NF subunits are depicted in sdceaticform. The code names of the anti-NF MAbs that labeled cortical LBs are indicated above or below the NF region containing the epitopes recognized by a given b. Abs to epitopes in the rod regions and the carboxy terminus bind in a phosphoylation-independen manner. MAbs to the multiphosphorylation repeat bind to phosphorylated epitopes. The exact binding site of RM0123 is notknown. Thisantibody binds to a ph orlaed epiope in the tail region of ~~~~~~NF-Moutsuide the multiphophoylation repeat. MAbs that bind to the multpopho-ylation repeat of both NF-M and NF-H bind to either

phosphorylated or dephosphorylated sites. AMAbs that are underlined indicate that these

MAbs also bind to epitopes to AD NFTs. 17,33 Several of these MAbs (ie, RMOs: 44, 57, 106, 189, 255; RMS12; TA66) were not included in previous publications on NFTs because they label NFTs only in ETOHINaCI-fixed tissues RMO 216, COOH, rod region; terminus, ROD,epitope; DEPHOS,carboVy MPR, NA 256,264 TAs1 NA NA dephosphorylated NA RMO 194 O NFH EH ROD T MPR T multiphosphorylation repeat; NA, no MAb available to this region; NEG, negative; NF-H, high-molecular-weightNFsubunit; NF-L, lou-molecular-weightNFsubunit;NF-M, middle-molecular-weightNFsubunit;NF-M + H, middle- and high-molecular-weight NF subunit; NH2, amino terninus; T, tail region; PHOS, phosphorylated epitope.

DEPHOS

-

DPi RMDO9 RfIDT2

62

Schmidt et al

AJP July 1991, Vol. 139, No. 1

A !l,

:'

;.;It,%-. !,- .1---

ip

.

It

-, ,

.,.

-

'-.

."

"

"

-OL.

Figure 7. Immunohistochemistry uas performed on adjacent hippocampus sections to show that RM032 does not stain NFTs, SPs, or NTs. Primary MAbs used were A: T14 a MAb to tau protein; B: an MAb to ubiquitin; C: RM032 an MAb to NF-M. All sections were counterstained with hematoAylin. Both T14 and anti-ubiquitin stained many NFTs, SPs, and ATs. There is no immunoreactivity in the hippocampal section labeled u'ith RM032 (C) although both RM032 and anti-ubiquitin labeled many LBs and pleomorphic inclusions in sections of several cortical areasfrom this AD patient. Space bar = 25R.

ularly shaped cortical LBs had similar ultrastructural features, occurred together in the same cortical laminae, and no case showed only one of these LB shapes. It is not known how LBs form. In the substantia nigra, however, almost all LBs appeared round or oval. This observation strongly suggests that irregularly shaped cortical LBs are not necessarily precursors of round and oval LBs or vice versa. The observation that the anti-ubiquitin MAb and RM032 immunostained the largest number of corti-

cal LBs, in comparison with other anti-NF or MAP2 MAbs, suggests that NF-M and ubiquitin are the dominant protein subunits present in LBs. The heterogeneous staining of LBs with these antibodies may reflect stages in the maturation or degradation of cortical LBs or conformational changes in the NF triplet proteins during their incorporation into cortical LBs. Our data indicate that epitopes distributed throughout each of the NF subunits, including phosphorylated and nonphosphorylated NF epitopes as well as MAP2 and ubiquitin epitopes, are present in cortical LBs just like the substantia nigra LBs of PD.16 Notably RM032, which stained the most cortical LBs, also immunostained the largest number of LBs in the substantia nigra of PD patients (Hill et al, unpublished data). The NF-M epitope recognized by RM032 appears to be the most ubiquitous constituent of all cortical LBs. Thus most cortical LBs may be composed of the NF-M subunit in a confirmation that does not permit its recognition by most other antNF-M MAbs. Alternatively a fragment of NF-M containing the RM032 epitope may be the major proteinaceous component of cortical LBs, but this notion leaves unexplained how this fragment assembles into the filaments that are dominant building blocks of cortical LBs. Finally an as yet unidentified protein that cross-reacts with RM032 may account for the fact that this MAb stained the largest number of cortical LBs. Because RM032 only detected a 1 70-kd band in Western blots of whole human brain homogenates, however, it is likely that RM032 is exclusively specific for human NF-M. Nevertheless we cannot exclude the possibility that a cross-reacting protein is present in the brain at a concentration too low to be detected by Western blotting. In this case the process leading to cortical LBs would selectively accumulate this unidentified protein to form these lesions. Peripheral nervous system LBs have been reported previously,51.52 but we have extended these observations by detecting NF- and ubiquitin-positive LBs in neurons of Auerbach's plexus, the adrenal medulla, and the dorsal root ganglion. The restricted number of LBs encountered in the PNS did not allow a complete epitope analysis to be performed. There is, however, a convincing immunohistochemical and ultrastructural similarity between cortical LBs and PNS LBs, and it is reasonable to consider them equivalent lesions. The variable occurrence of LBs in the PNS of the cases studied here together with their irregular distribution in different PNS sites (Table 4) make it unlikely that biopsy of these PNS regions will be of diagnostic utility. The widespread occurrence of LBs in the CNS and PNS of demented individuals and their paucity in controls, however, suggests that disruption of NF metabolism or transport may be a molecular substrate for neuron dysfunction and degeneration in these conditions.


63


Table 4. Location of LBs in PNS Sites

Colon

Esophagus

Trigem.

Diagnosis

DRG

ganglion

Adrenal

(lower)

Stomach

Ileum

(ascending)

AD/PD AD/PD AD/PD PD/DEM PD/DEM PD/DEM DLBD

0 0 0 0 0 0 1

NA NA NA 0 0 0 0

0 1 2 0 2 0 3

1 0 2 NA 4 NA 11

0 0 0 NA 0 NA NA

2 0 0 2 0 5 0

0 0 2 0 1 15 2

NA, not applicable. This table includes a subset of the patients listed in Table 2 that had PNS LBs. The number and location of these PNS LBs are summarized here. Abbreviations are shown in the legend to Table 1.

Acknowledgments The authors thank Ms. C. Page and Ms. A. O'Brien for technical assistance. Drs. G. Gottlieb, M. Grossman, R. C. Gur, H. Hurtig, and G. G. Pietra, as well as Mr. J. DiRienzi and Mr. G. Davis, aided in the collection of postmortem tissues. Mrs. H. Comstock of the Philadelphia Alzheimer's Disease and Related Disorder Association, Inc., and the families of the patients studied here made our work possible through their generous efforts to foster research.

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