disease-associated alz-50 immunoreactivity quantified in

American Journal of Pathology, Vol. 141, No. 6, December 1992Copyright X) American Association ofPathologists

Immunohistochemical and HistopathologicCorrelates of Alzheimer'sDisease-associated Alz-50Immunoreactivity Quantified inHomogenates of Cerebral Tissue

Suzanne M. de la Monte,* Rachel A. Spratt,*Jonathan Chong,t Hossein A. Ghanbari,t andJack R. Wands*From the Massachusetts General Hospital Alzheimer's DiseaseResearch and Cancer Centers, Divisions ofNeuropathologyand Medical Services,* Massachusetts General Hospital,Harvard Medical School, Boston, Massachusetts, and theResearch and Development Division,t NeuropsychiatricMarkers Research and Development, Abbott Laboratories,Abbott Park, Illinois

Alz-50 is a monoclonal antibody that immunoreactswith neurofibrillary tangles and neurites in brainswith Alzheimer's disease (AD). In addition, the levelsof Alz-50 immunoreactivity in brain, measured byeither enzyme-linked immunosorbent assay or ALZ-enzyme-linked immunosorbant assay (EIA), are in-creased in AD relative to age-matched controls. Thecurrent study compares the distribution and extent

ofAlz-50 immunostaining with quantified levels ofAlz-50 immunoreactivity measured in adjacent fro-zen blocks oftissue byALZ-EIA The brain tissue stud-ied was obtained from individuals with AD, AD +

Doum's syndrome (AD + DN), Parkinson's diseasewith dementia (PD), orAD + PD, andfrom nonde-mented aged controls. In AD, AD + DN, and AD +

PD, there were significantly higher densities of Alz-5S-immunoreactive (AFI) neurons, more abundantdiffuse AFI neurites, and higher ALZ-EA values thanin aged controls. In PD, the overall mean density ofAFI neurons was significantly lower than in AD andAD + DN, butAFI neurites were as abundant as theywere in brains with an AD diagnosis. However, PDwas readily distinguishedfrom AD andAD + DN bysignificantly lower mean ALZ-EIA values, and signif-icantly lower densities of neurofilament-immuno-reactive AD lesions. Multiple-regression analysisdemonstrated significant correlations between ALZ-FHA levels and the severity of AD lesions, and the

density ofAFI neurites but not with the density ofAFIneurons. Therefore, ALZ-EIA levels may representonly a portion of the Alz-50 immunoreactivity de-tectable by immunohistochemical staining. (Am JPathol 1992, 141:1459-1469)

The Alz-50 monoclonal antibody immunoreacts with a68-kd protein (A68)1-5 that is 15 to 50 times more abun-dant in brains with Alzheimer's disease (AD) than inbrains from aged controls.1 2 A68 probably represents amodified form of phosphorylated tau,5-10 but the mech-anism and significance of the molecular alteration areunknown. In histologic sections, Alz-50 immunoreactivityis colocalized with neurofibrillary tangles and dystrophicneurites,1'2'5,11'12 the major filamentous lesions corre-lated with dementia in AD.1>20 By immunoelectron mi-croscopy, Alz-50 binds to paired helical and straight fil-aments associated with AD lesions.52122 Consequently,the degree of Alz-50 immunostaining in cerebral tissue isgreater in AD than it is in aged controls.1 2 IncreasedAlz-50 immunoreactivity is not specific for AD, however,because it is observed also in filamentous intraneuronalinclusions encountered in other neurodegenerative dis-eases such as Pick's disease, progressive supranuclearpalsy, and Parkinson-dementia complex of Guam, aswell as in the setting of neuronal ceroid lipofuscinosis, ie,Kufs' disease.222-25 Moreover, Alz-50 immunoreactivityis detectable in histologically normal-appearing neurons,ie, neurons without neurofibrillary tangles,1' 211'21 sug-gesting that the antibody binding is not restricted to the

Supported by Kl 1-AG00425 from the National Institute on Aging, RO1-NS29793 from the National Institute of Neurological Disorders and Stroke,the American Health Assistance Foundation, and RO1-AA-02666, CA35711, AA 08169, and HD 20469 from the National Institutes of Health.J.R.W. is the recipient of a Research Scientist Award AA-00048.

Accepted for publication June 16, 1992.Address reprint requests to Dr. Suzanne M. de la Monte, MGH Can-

cer Center, MGH East, 149 13th St., Charlestown, MA 02129.

1459

1460 de la Monte et alAJP December 1992, Vol. 141, No. 6

filamentous aggregates that typify AD neurodegenera-tion. Despite its lack of disease specificity with respect tolabeling neurodegenerative filamentous lesions, using anenzyme-linked immunosorbent assay, the quantified lev-els of Alz-50 immunoreactivity in cerebral tissue1' 2 andcerebrospinal fluid2 were demonstrated to be signifi-cantly elevated in AD compared with both aged and dis-ease controls. The observations with respect to cerebraltissue have since been confirmed26 using a more spe-cific assay, the ALZ-EIA.27

Our objective is to understand the molecular basis ofneuronal degeneration in AD. To this end we have beenexploring the nature and distribution of accumulated im-munoreactive materials in AD because they may reflectoverexpression of a particular gene or aberrant process-ing of a gene product. For example, increased Alz-50immunoreactivity in AD might be caused by increasedtranscription of the gene, or the accumulation of insolubleantigenic material associated with filamentous neurode-generative lesions. To begin to answer this question, thecurrent study was designed to examine the correlationbetween the distribution of Alz-50 immunohistochemicalstaining and the quantified levels of Alz-50 immunoreac-tivity in cerebral tissue with different degrees of severity ofAD lesions, and in normal aging.

Methods

Source of Tissue

Postmortem brain tissue was obtained from individualswith histopathologically diagnosed AD, Parkinson's dis-ease (PD), or Alzheimer's plus Parkinson's disease (AD+ PD). These patients were clinically demented and dur-ing life had been serially evaluated with neurologic ex-aminations and neuropsychiatric testing at the Massa-chusetts General Hospital (MGH) Alzheimer's DiseaseResearch Center (ADRC). Brain tissue was obtained alsofrom individuals with Down's syndrome and Alzheimer'sdisease lesions (AD + DN), and from aged controls with-out clinically manifested neurologic or neurosurgical dis-ease. The individuals with AD + DN had been chronicresidents at the Fernald State School in Waltham, Mas-sachusetts, and the aged controls were patients who hadbeen regularly evaluated at the MGH. The brain tissuewas harvested within 18 hours of death according to theMGH-ADRC protocol.28 Histopathologic diagnoses wereestablished using paraffin-embedded tissue sectionsstained with luxol fast blue, hematoxylin and eosin, Biel-schowsky silver impregnation, and Congo red. The diag-nosis of AD was rendered using threshold criteria estab-lished by the NIA-NINCDS group.' In addition, all brains

diagnosed with AD, including those with AD + DN andAD + PD, had neocortical neurofibrillary tangles.

The diagnosis of PD was based on neuronal loss, gli-osis, and intraneuronal Lewy body inclusions in the sub-stantia nigra, locus coeruleus, and dorsal motor nucleusof the vagus nerve. The brains that were classified as PDalso contained plaques and neurofibrillary tangles, buttheir densities were insufficient to render the diagnosis ofAD + PD. The diagnosis of AD + PD was made whendiagnostic criteria for both diseases were present in thesame brain. Patients in both the PD and AD + PD groupshad the clinical diagnosis of Parkinson's disease formany years, but within the last 5 years of life they devel-oped dementia, which was thought to represent super-imposed Alzheimer's disease. Bielschowsky silver stain-ing and immunohistochemical staining with polyclonalantibodies to ubiquitin (Biomeda Corp., Foster City, CA)or with monoclonal antibodies to neurofilament (SMI 31and 34) failed to disclose widespread Lewy bodies insections of the cerebral cortex from patients in the PD orAD + PD groups; therefore, these cases were not clas-sified as diffuse Lewy body disease. Although the ubiq-uitin antibody we used might not have been as sensitiveas the one used in other studies of Parkinson's dis-ease,3032 we were able to detect cortical Lewy bodies inunequivocal cases of diffuse Lewy body disease.

Immunohistochemical Studies

Formalin-fixed, paraffin-embedded sections (8 IL thick)from Brodmann areas 24, 11, and 4 in the frontal lobe,Area 21, the CAl + CA2 regions of the hippocampalformation, and the amygdaloid nuclear complex in thetemporal lobe, area 40 in the parietal lobe, area 17 in theoccipital lobe, the putamen, globus pallidus, and the thal-amus (level of lateral geniculate body) were immuno-stained with Alz-50 monoclonal antibody derived fromhybridoma supernatant.1 The antibody was used diluted1:100 in phosphate-buffered saline (10 mmolA sodiumphosphate, 0.15 molA sodium chloride, pH 7.35) contain-ing 1% bovine serum albumin (PBS-BSA). This dilution ishigher than that used by many other groups2.8,21,3336;however, the commercial detection assay we employedis highly sensitive and routinely permits use of fivefold to10-fold greater dilutions of the primary antibody com-pared with other protocols. Moreover, in other studies inwhich little or no nonfat dry milk was used as a blockingagent, and a detection protocol similar to the one weused was employed, Alz-50 immunoreactivity also wasdetected with the same monoclonal antibody diluted 1:100 rather than 1:5 or 1:1 0.37,38

Before immunostaining, the sections were de-waxedin xylenes and hydrated through graded alcohol solu-

Alz-50 Immunoreactivity in AD 1461AJP Decemnber 1992, Vol. 141, No. 6

tions. Nonspecific binding was blocked by incubating thesections with 1% nonimmune horse serum for 30 min-utes. The sections were incubated overnight at 4°C withthe Alz-50 or other primary antibody. Endogenous per-oxidase activity was blocked by treating the sections with0.6% hydrogen peroxide in methanol for 30 minutes. Im-munoreactivity was detected by the avidin-biotin horse-radish peroxidase method (Vector ABC Elite Kit; VectorLaboratories, Burlingame, CA) following the manufactur-er's protocol, and with 0.05% 3-3' diaminobenzidine(Sigma) as the chromogen. The sections were counter-stained with hematoxylin, dehydrated in graded alcoholsolutions, cleared in xylenes, and preserved under cov-erglass with Permount (Fisher Scientific Products, NewYork, NY). All incubations, except the one with primaryantibody, were carried out at room temperature. Betweensteps, the sections were washed with three changes ofPBS (5 minutes). All serologic agents were diluted inPBS-BSA. To determine the density of neurofibrillary tan-gles, adjacent sections were immunostained with mono-clonal antibodies to the phosphorylated middle and highmolecular weight neurofilament proteins (SMI 31 and SMI34; Stemnberg and Meyers, Inc., Baltimore, MD).3941 Inaddition, adjacent sections were immunostained withmonoclonal antibodies to glial fibrillary acidic protein(Dako Corp., Carpinteria, CA) as a positive control, andwith monoclonal antibodies to Dengue virus as a nega-tive control. For each antibody, all sections were pro-cessed together under identical conditions.

Analysis of Immunoreactivity

The sections were examined by light microscopy to de-termine the regional distribution of ALZ-50-immunore-active (AFI) neurons and neurites. The density of AFI neu-

rons was determined by counting the number of labeledneurons present in 40 adjacent neuron-containing fieldsat a magnification of 400x using a standard ocular grid.To do this, the full thickness of cortex or gray matter struc-ture was surveyed using a mechanical microscopestage, beginning the enumeration in layer 1 at the crest ofa gyrus and proceeding systematically toward layer 6,then across, upward toward layer 1, across, then down-ward again, etc., always keeping the horizontal progres-sion unidirectional. The densities of intraneuronal neuro-

filament-immunoreactive neurofibrillary tangles were enu-

merated in the same manner. The density of AFI neuriteswas assessed qualitatively as high (>7500/mm2), inter-mediate (>2500/mm2), low (>25/mm2), or absent (<25/mm2, but usually 0). The scores were based on the max-imum density of AFI neurites present in the microscopicfields examined to determine the density of AFI neurons.

The immunohistochemical staining and analysis were

performed at the Massachusetts General Hospital withthe observers blinded to the established histopathologicdiagnoses.

Quantitative Assessment of Alz-50Immunoreactivity by ALZ-EIA

Unfixed snap-frozen blocks of tissue immediately adja-cent to the paraffin-embedded blocks used for histopath-ologic and immunohistochemical studies were assayedfor levels of AD-associated Alz-50 immunoreactivity us-ing ALZ-EIA.27 Tissue extracts were prepared by pulver-izing the frozen tissue on dry ice, and then homogenizingthe fragments in five volumes of PBS containing 0.02%sodium azide using a Polytron (Brinkmann InstrumentsInc., Westbury, NY). The homogenates were centrifugedat 1 0,000g for 20 minutes at 40C, and the supernatantfractions were used to quantify Alz-50 immunoreactivityand protein concentration. Aliquots of these extracts weresent frozen under code to Abbott Laboratories, where theALZ-EIA was performed. The results were forwarded tothe Massachusetts General Hospital for analysis. The lev-els of Alz-50 immunoreactivity were expressed as absor-bency per milligram total protein. Protein concentrationwas measured by the Lowry colorimetric assay.42

Statistical Analysis

The data were analyzed by comparing the groups withrespect to the densities of AFI neurons and neurites, andthe levels of PBS-extracted Alz-50 immunoreactivity mea-sured by the ALZ-EIA. The densities of AFI neurons andneurofilament-immunoreactive neurofibrillary tangles (ad-jacent sections) and ALZ-EIA values were expressed asmean ± standard error of the mean, and intergroup com-parisons were made using analysis of variance. Whensignificant F-ratios were obtained, Fisher LSD and Dun-can a posteriori tests were used to determine whichgroups were significantly different from the others. Inter-group comparisons with respect to the frequency distri-butions of high-density, intermediate-density, low-density, and absent AFI neurites were made using cross-tabulation chi-square analyses. The specific groupsresponsible for the significant differences were deter-mined from the chi-square values obtained for individualcells. Multivariate regression analysis was used to di-vulge significant correlations between ALZ-EIA valuesand the densities of Alz-50-immunoreactive neurons,neurites, senile plaques, and neurofibrillary tangles. Thedata were analyzed using the Number Cruncher Statisti-cal System, version 5.01 (Dr. Jerry L. Hintze, Kaysville,


UT) interfaced with an IBM-compatible personal com-puter.

Results

Population Profile

The brain tissue used in this study was obtained fromindividuals with AD (n = 20), AD + DN (n = 5), AD +PD (n = 7), and PD (n = 6), and from 11 nondementedaged controls. The groups were similar with respect toage and gender distributions, except that the mean ageof the AD + DN group was 10 to 14 years lower thanthose of the other groups (P < 0.001), and females con-stituted a significantly greater proportion of the patients inthe AD group compared with the other groups (P <0.001) (Table 1).

In total, 208 paraffin-embedded blocks of tissue (99AD, 32 AD + DN, 18 AD + PD, 1 1 PD, and 48 control)were examined for Alz-50 immunoreactivity by immuno-histochemistry. Another 178 snap-frozen blocks (64 AD,seven AD + DN, nine AD + PD, 20 PD, 78 control) wereassayed for levels of Alz-50 immunoreactivity by ALZ-EIA. Discrepancies between the number of paraffin andfrozen blocks studied were due to unavailability of somefrozen blocks in all groups, and the inclusion of blocks ofdeep frontal white matter from AD and control cases foranalysis by ALZ-EIA. In addition, for the control group,bilateral and duplicate samples of frozen tissue wereevaluated by ALZ-EIA to help strengthen the significanceof the findings.

Tissue blocks representing Brodmann areas 11, 24,21, and 40, and the amygdala were available from allgroups. For areas 4,17, and the hippocampal formation,tissue blocks were only available from the AD, AD + DN,and control groups. Sections of the globus pallidus, puta-men, and thalamus from AD, PD, and control brains alsowere surveyed for Alz-50 immunoreactivity, but cellularlabeling was sparse and difficult to interpret given thesingle plane of section through several different nucleargroups. Therefore, data derived from these regions were

Table 1. Demographic Profile

Number Ageof (mean

Diagnosis cases + SEM) Gender

Alzheimer's disease 20 75.5 ± 2.5 5M; 15FAlzheimer's & Down's

syndrome 5 64.5 8.1 3M; 2FAlzheimer's &

Parkinson'sdisease 7 75.2 ± 10.4 5M; 2F

Parkinson's disease 6 78.5 ± 3.9 5M; 1FAged controls 11 77.3 ± 7.9 1OM; 1F

excluded from the statistical analyses. Several blockswere missing from all groups, but particularly from AD +PD and PD, because the tissue had been exhausted inprior studies. Despite these limitations, the study provedrevealing because a large number of immunostainedsections from different regions of brain were analyzed indetail, and the patterns of immunohistochemical stainingwere compared with the quantitative levels Alz-50 immu-noreactivity measured by ALZ-EIA.

Immunohistochemical Studies

Distribution of Alz-50 Immunoreactivity

Alz-50 immunoreactivity was present in neuronalperikarya and neurites of cerebral tissue (Figure 1).Perikaryal labeling was either granular or dense and fibril-lar. Granular immunoreactivity was observed in brainsections from all groups, including control. Fibrillar label-ing was observed in all groups except control, and fibril-lar labeling frequently appeared to be associated withneurofibrillary tangles. Extraneuronal neurofibrillary tan-gles were not immunoreactive with Alz-50, however. InAD and AD + DN, most of the perikaryal labeling wasfibrillar, whereas in AD + PD, PD, and controls, most orall of the perikaryal labeling was granular. Neuritic label-ing was observed both at the periphery of plaques andscattered throughout the neuropil and white matter. Al-though AD filamentous lesions were detected by Alz-50,adjacent sections immunostained with antibodies to neu-rofilament or silver stained by the Bielschowsky methodshowed higher densities of plaques, neurofibrillary tan-gles, and neurites. Histologic sections from all cases ex-hibited immunoreactivity for neurofilament and glial fibril-lary acidic protein, and none exhibited immunoreactivitywith the negative control Dengue virus antibody, or whenno primary antibody was used.

Density of Alz-50 Immunoreactive Neurons:Between-Group Analyses

Alz-50-immunoreactive neurons were detected inmost of the histologic sections (139 of 208, 67%) exam-ined, but their densities varied significantly among thegroups (P < 0.001). With data from all regions combined,AFI neurons were significantly more abundant in AD, AD+ DN, and AD + PD than in aged control brains (P <0.001, P < 0.001, P < 0.01, respectively). The highestmean density was observed in AD + DN (P < 0.001relative to all other groups) (Figure 2). For the AD, AD +PD, and PD groups, the standard errors of the meansoverlapped, and therefore, the differences among thesegroups were not statistically significant. For nearly all re-gions, the highest mean densities of AFI neurons were

AIz-50 Immunoreactivity in AD 1463AJP December 1992, Vol. 141, No. 6

.. *.; 4 ' 2..-:s::.-+!';-'w' P'V tgz-X <,-

Figure 1. Alz-50 immunohistocbemisty. Aged control with negative staining (al) and granular immunoreactivity in neuronalperikarya(a2). Fibrillar immunoreactivity in neuronalperikarya in AD (a3). Note negative neuron in samefield (arrow). Neuritic labeling associatedwith amyloid-containing plaques (b1, arrows), and scatered in the neuropil with moderate (b2) or high (b3) densities.

observed in AD + DN, followed by AD or AD + PD. Incontrol brains, AFI neurons were detected but they wereextremely rare, with mean values nearly always ap-proaching zero, except in the CAl + CA2 region of thehippocampal formation.

In the frontal lobe as a whole, and in Area 11 specif-ically, the mean densities of AFI neurons in AD, AD +

DN, and AD + PD were significantly higher than in PD (P< 0.005, P < 0.001, and P < 0.05, respectively) andaged controls (P < 0.005, P < 0.001, and P < 0.01,respectively) (Figures 2, 3). In area 24, the mean densi-ties of AFI neurons were significantly greater in AD andAD + DN than in controls (both P < 0.05), and in AD +DN relative to AD and PD (both P < 0.05). For area 4 inthe frontal lobe, AFI neurons were more abundant in AD

than in AD + DN (P < 0.01) and controls (P < 0.005).Sections from area 4 were unavailable from the AD + PDand PD cases. In area 40 of the parietal lobe, AFI neuronswere significantly more abundant in AD and AD + DNcompared with AD + PD and controls (all P < 0.05).

In the temporal lobe, with data from Area 21 and theCAl + CA2 region of the hippocampal formation com-

bined, the mean densities of AFI neurons overlappedamong all groups except AD + DN, which exhibited themost abundant labeling (P < 0.001). With respect to area21 itself, the densities of AFI neurons in AD, AD + PD,and PD were similar, and all were significantly greaterthan in control brains (P < 0.01) in which they were es-

sentially absent. In the CAl + CA2 region of the hippoc-ampal formation, as well as in area 17 of the occipital

...0.4


ALZ-S IABELEDNEURLNS

~~~~~~~~~~~~~~AD.DAD.PDi

l-1-m 0 A]- *s- V

v 13~~~~~~~~~~~~ PD

561t4 Lit(tALLfI6tm FROTA PRAL THOA

Figure 2. Comparison of the mean densities (±SEM) of Alz-50immunoreactive (AFI) neurons (top) and intracellular neurofila-ment immunoreactive neuroflbrillaiy tangles (bottom) in adja-cent immunostained sections. Black dots indicate missing values.With respect to AFI neurons, statistically significant differenceswere asfollows (see te-xtfor P-values): all regions-between AD, AD+ DN, or AD + PD and aged controls; frontal cortex-betweenAD, AD + DN, or AD + PD and PD or aged controls; parietalcortex-between AD orAD + DN andAD + PD or aged controls;temporal cortex-between AD + DN and all otber groups; occip-ital cortex-between AD orAD + DN and age controls. The meandensities ofneuroflamnent-immunoreactive neurofibrtllary tanglesin each region examined were significantly greater in AD orAD +DN than in AD + PD, PD, and aged controls.

cortex, the densities of AFI neurons were significantlygreater in AD and AD + DN than in controls (both P <0.01 and P < 0.05, respectively). Sections from the CAl+ CA2 region and area 17 were not available from theAD + PD and PD groups. In the amygdala, in contrast toall other regions, the mean densities of AFI neurons weresignificantly greater in AD + PD and PD than in controls(P < 0.001 and P < 0.01, respectively), and the differ-ence between AD and control was not statistically signif-icant. In the globus pallidus, putamen, and thalamus onlyrare scattered AFI neurons were observed in brains withAD, AD + PD, or PD.

In contrast to the considerable overlap observedamong the disease groups with respect to density of AFIneurons, AD and AD + DN were unambiguously distin-guished from AD + PD, PD, and control by the strikinglyhigher densities of neurofilament-immunoreactive neuro-fibrillary tangles (P < 0.001) (Figure 2). Although themean densities of neurofibrillary tangles in AD and AD +DN varied widely, the differences from the other groupswere highly statistically significant for all regions (P <0.001). Neurofibrillary tangles were enumerated in histo-logic sections immunostained with the SMI 31 and SMI34 monoclonal antibodies to high and middle molecularweight phosphorylated subunits of neurofilament.39-41The sections immunostained for neurofilament were im-mediately adjacent to those immunostained with Alz-50.The densities of neurofilament-immunoreactive and Biel-schowsky silver-impregnated neurofibrillary tangles werecomparable.

* AD

E AD+DNE AD+Pi)O PDo COhTROL

01 nf1:L 1 MollI-X_AREA CAI + ('A2 ARI'A 17 AMYGiUAI.A

Figure 3. Regional between-group differences in mean density(±SEM) of Alz-50 immunoreactive (AFI) neurons. Black dots in-dicate missing values. Significant differences: area 11-betweenAD, AD + DN, or AD + PD and PD or aged controls; area 24-between AD or AD + DN and aged controls, and between AD +DN and AD or PD; area 4-between AD and AD + DN or agedcontrols; area 40-between AD or AD + DN and AD + PD oraged controls; area 21-between aged controls and all othergroups, and between AD + DN and all other groups; CAl +CA2-between AD or AD + DN and aged controls; area 17-between AD orAD + DN and aged controls; amzygdala-betweenAD + PD or PD and AD or aged controls.

Regional Variation in the Density ofAlz-50-Immunoreactive Neurons:Within-Group Analyses

In AD, the highest mean densities of AFI neurons wereobserved in Brodmann area 11 and area 4. Intermediatedensities were measured in areas 24, 21, 40, and 17, andin the CAl + CA2 region of the hippocampal formation,and low densities were observed in the amygdala (Figure3). In AD + DN, high densities of AFI neurons werepresent in five of the eight regions studied (Brodmannareas 11, 24, 40, and 21, and the CAl + CA2 region ofthe hippocampal formation), and therefore AFI neuronswere diffusely more abundant than in AD. In area 4 andarea 17, the mean densities of AFI neurons in AD + DNwere 50% lower than in other regions, but neverthelesswere intermediate in density compared with the findingsin groups with an AD diagnosis. In AD + PD, like AD, thehighest density of AFI neurons was observed in area 11of the frontal lobe, and intermediate densities were ob-served in area 40, area 21, and the amygdala. In PD, in

.1 -I

AIz-50 Immunoreactivity in AD 1465AJP December 1992, Vol. 141, No. 6

contrast to brains with AD diagnosis, AFI neurons werevirtually absent in area 1 1 of the frontal lobe, and thedensities were highest in area 21, area 24, and theamygdala. Control brains contained relatively few AFIneurons throughout most regions of brain, and they didnot exhibit appreciable regional variation akin to that ob-served in disease brains.

Alz-50 Immunoreactivity in Neurites

Alz-50-immunoreactive neurites were observed at theperiphery of plaques and scattered within the neuropil inall groups. Only the diffuse neuropil component was con-sidered in the qualitative assessment of neuritic density,however. The data from all regions were analyzed to-gether because the regional variation was not statisticallysignificant. Alz-50-immunoreactive neurites were de-tected in 90% or more of the AD, AD + DN, AD + PD,and PD sections, compared with only 31% of control (P <0.001) (Figure 4, bottom). Moreover, in 62% of AD, 69%of AD + DN, 61% of AD + PD, and 73% of PD sections,the density of AFI neurites was moderate or high,whereas in controls, none of the sections exhibited highdensities, and only 8% exhibited moderate densities ofAFI neurites. Instead, in control brain sections, AFI neu-rites were mainly either sparsely distributed (23%) or ab-sent (69%).

ALZ-EIA Levels in Brain Tissue HomogenatesThere were significant intergroup differences in the

overall mean ALZ-EIA levels (all regions combined ex-

Abundant

Moderate

Few

None

DENS/YOFAIS-5 LABELED NEUR17E

-11S.. 11 1111

I~~I~ -

AD AD+DN AD+PD PD ControlFigure 4. Between-group differences in the density of diffuse Alz-50 immunoreactive dystrophic neurites. 7he density ofAFI neuriteswas assessed qualitatively as high (>7500/mm), intermediate(>2500/mmn), low (>25/mmn), or absent (<25/mm2, but usually0). The scores were based on the maximum density ofAFI neuritespresent in a representative area of the section. Data from eightdifferent cerebral regions were combined. Each dot represents thescore assigned to an individual slide. The distrbution densities ofAFI neurites were similaramong the AD, AD + DN, AD + PD, andPD groups, in which thepercentages ofsections exhibiting moder-ate or high densities ofAFI neurites were significantly greater thanin the aged control group.

cept for white matter; P < 0.001) (Figure 5). The highestmean values were measured in AD and AD + DN, fol-lowed by AD + PD. The lowest was in the control group.In PD, the mean ALZ-EIA was similar to control, but thestandard error of the mean was high because of overlapof individual cases with AD + PD. The overall mean ALZ-EIA levels were significantly higher in AD (P < 0.005), AD+ DN (P < 0.005), and AD + PD (P < 0.05) than in agedcontrols, and in AD (P < 0.005) and AD + DN (P <0.005) compared with PD.

Frontal Cortex

In the frontal lobe, the mean ALZ-EIA level in AD wassignificantly higher than in PD (P < 0.01) and aged con-trols (P < 0.005). In contrast, in AD + DN, AD + PD, andPD the mean ALZ-EIA levels were not significantly differ-ent from control because of wide individual variationswithin these groups. With respect to specific regions, in

ALZ-EIA

5)

A

0.4

0.3-

Q4.0 0.2.

0.1 .

* AD

M AD.DN* AD+PDo PD

M CONTROL

MIA ---- th.***-eTEMPORAL AMYUDALA WHITE MATMER

Figure 5. Between-group differences in mean levels (+SEM) ofAlz-50 immunoreactivity measured in PBS-extracts ofcerebral tis-sue using ALZ-EIA Black dots indicate missing values. Tbe signif-icant between-group differences were as follows: all regions (ex-cluding white matter)-between AD, AD + DN, or AD + PD andaged controls, and between AD or AD + DN and PD; frontalcortex-between AD and PD or aged controls; parietal cortex-between AD + DN and PD or aged controls; temporal cortex-betweenAD orAD + DNand aged controls, and between AD andPD; amygdala andfrontal white matter-hetween AD and con-trols.

0 __ __ M I _ =.n-L--..I


area 11, the AlZ-EIA levels in AD were significantlyhigher than in PD and control (both P < 0.05). In area 24,the mean levels was also significantly higher in AD than incontrol (P < 0.05). For area 4, the number of samplesanalyzed was insufficient for statistical analysis.

Parietal Cortex

In area 40 of the parietal lobe, the highest mean ALZ-EIA value was observed in AD + DN. Next were AD andAD + PD, in which the mean levels were similar. Thelowest mean ALZ-EIA levels were observed in PD andcontrol. The only statistically significant differences oc-curred between AD + DN and PD, and between AD +DN and control (both P < 0.05).

Temporal Cortex

In the temporal lobe, the highest mean ALZ-EIA levelswere observed in AD and AD + DN, followed by AD +PD. In PD and aged controls, the mean levels were sim-ilar and lowest among the groups. For AD and AD + DN,the mean ALZ-EIA levels were significantly higher than inthe control group (P < 0.01 and P < 0.05, respectively).Also, the mean AlZ-EIA was significantly higher in ADthan in PD (P < 0.05).

impregnated neurofibrillary tangles using multiple regres-sion analysis.

ALZ-EIA Measurements

The ALZ-EIA levels were significantly correlated withthe density of AFI neurites (r = 0.51, P < 0.005), and theabundance of neurofibrillary tangles (r = 0.63, P <

0.001). The ALZ-EIA values were not correlated with den-sity of AFI neuronal perikarya (r = 0.007, not significant).

Diagnoses

The diagnosis of Alzheimer's disease, either alone oras a component of Down's syndrome or Parkinson's dis-ease, was significantly correlated with ALZ-EIA levels (r= 0.64, P < 0.005), the density of AFI neurites (r = 0.69,P < 0.005), and the density of neurofibrillary tangles (r =0.69, P < 0.01), but not the density of AFI neurons(perikaryal labeling). Using stepwise multivariate regres-sion, the significant independent predictors of AD diag-nosis were AlZ-EIA value, density of neurofibrillary tan-gles, and density of AFI neurites (all P < 0.05). The ALZ-EIA values were not significantly correlated with thediagnosis of PD.

DiscussionOther Regions

Statistical analysis of data derived from the occipitalcortex was precluded by the small number of samplesavailable for study. In AD, the mean ALZ-EIA levels in theamygdala and white matter were threefold to fivefoldlower than in cortical regions. Equally low ALZ-EIA levelswere observed in the globus pallidus, putamen, and thal-amus, but these data were not analyzed statistically be-cause of small sample sizes. In PD and control, the meanALZ-EIA levels in the amygdala were similar to the levelsin other regions. There was virtually no Alz-50 immunore-activity detectable by ALZ-EIA in control frontal whitematter. The mean ALZ-EIA levels in AD amygdala andfrontal white matter were significantly higher than in con-trols (both P < 0.05).

Correlates of Alz-50 Immunoreactivity

Immunohistochemical Labeling

There were no significant correlations observed be-tween the densities of AFI neuronal cell bodies and thedensities of AFI neurites, neurofilament-immunoreactiveneurofibrillary tangles, or Bielschowsky silver-

Distribution of Alz-50 Immunoreactivity inImmunostained Tissue Sections

Alz-50 immunoreactivity was observed in neuronalperikarya, neuropil and white matter fibers, and neurites,as described previously.' 25,11'12 Neuronal perikaryal im-munoreactivity was characterized as either granular orfibrillar in nature, but fibrillar labeling was more prevalentin brains with AD lesions. In most brains with AD or PDlesions, variable densities of both fine and coarse irreg-ular AFI neurites were observed in the neuropil and whitematter, as well as at the periphery of plaques, corre-sponding with the distribution of neurofilament-immunoreactive and Bielschowsky silver-stained neu-rites in adjacent sections. This is consistent with results ofprevious studies in which co-localization of Alz-50 immu-noreactivity with thioflavin S or Bielschowsky silver-stained AD lesions was demonstrated. 1133,34,36,37,43 44The densities of AFI neuritic plaques and neuropil neu-rites were lower, however, than those detected by immu-nostaining for middle and high molecular weight phos-phorylated neurofilament proteins or by Bielschowsky sil-ver impregnation. Moreover, clearly recognizable AFIneurofibrillary tangles were less abundant than neurofila-ment-immunoreactive or silver-stained neurofibrillary tan-

Alz-50 Immunoreactivity in AD 1467AJP December 1992, Vol. 141, No. 6

gles in adjacent sections. Discordances between thiofla-vin S staining and Alz-50 immunoreactivity in neurofibril-lary tangles have been reported previously.1 2'11 Potentialexplanations for underdetecting AD lesions by Alz-50 in-clude masking of the antigenic epitope by tissue fixation,as occurs with other filament-associated immunoreactiv-ties.45 Alternatively, Alz-50 immunoreactivity may bepresent in only a subpopulation of neurofibrillary tanglesand neurites in AD.11

Differences in the Quality ofImmunoreactivity and Density of AFINeurons in AD and AD + DN Comparedwith Other Groups

The finding of more abundant AFI neurons and neuritesin AD and AD + DN compared with age-matched con-trols is consistent with previous reports.1' 2 1 The findingof higher densities of AFI neurites in brains with AD le-sions is consistent with the previously demonstrated cor-relation between neuritic pathology and AD demen-tia 13'14'16'17'19'20 In AD and AD + DN, however, one ofthe most distinctive findings was that the neuronalperikaryal immunoreactivity was predominantly fibrillar,whereas in the control and PD groups, the immunoreac-tivity was largely or exclusively granular in nature. In AD+ PD, the mean densities of AFI neurons in differentbrain regions were similar to AD, but like PD and controls,the perikaryal labeling was predominantly granular ratherthan fibrillar. Of note is that the AD + PD brains includedin this study had less extensive AD lesions than did theAD and AD + DN brains (Figure 2), and with respect toPD, although AD lesions were detected, they were mildand insufficient to render the diagnosis of AD + PD. Dif-fuse Lewy body disease was not diagnosed in either theAD + PD or PD groups because widespread corticalLewy bodies were not detected in adjacent sections im-munostained for ubiquitin or neurofilament.

Altogether, it appears that the fibrillar type of Alz-50immunoreactivity in neuronal perikarya reflects or paral-lels the presence of substantial or advanced AD neuro-degeneration, which is detectable by other stainingmethods. In contrast, the granular type of labeling, whichwas prevalent in PD and AD + PD, but also detected incontrol brains, might represent an early neurodegenera-tive change, or perhaps a nonspecific alteration in neu-rons. Granular type Alz-50 immunoreactivity in neuronalperikarya in normal adult human brains has been de-scribed previously.46 In PD, the presence of scatteredAFI neurons with fibrillar labeling may suggest impendingprogression toward AD + PD.11 Increased Alz-50 immu-noreactivity in neuronal perikarya must reflect a processother than, or in addition to, the pathologic accumulation

of paired helical and straight filaments because neuronsthat lacked neurofibrillary tangles were also sometimeslabeled by the antibody, particularly in control and PDbrains. There is still no definitive explanation for this phe-nomenon, but the results of previous investigations sug-gest that increased Alz-50 immunoreactivity may markneurons destined to develop neurofibrillary tangles.11 Incircumstances unrelated to AD, such as development,increased Alz-50 immunoreactivity has been observed inneurons programmed to die.3'5 8

Overlap in the Patterns of Alz-50Immunostaining Among AD, AD + PD,and PD

Alzheimer's disease, AD + PD, and PD could not beconsistently distinguished from one another on the basisof the density of AFI neurons and relative abundance ofAFI neurites in the neuropil. As discussed, however, fibril-lar perikaryal immunoreactivity was a characteristic fea-ture of brains with advanced AD lesions and not of thosewith PD lesions. In contrast, high or intermediate densitiesof AFI neurites were a feature of brains with well-established AD or PD lesions. Together with previous ob-servations of increased Alz-50 immunoreactivity in brainswith Pick's disease and progressive supranuclearpalsy,2'22'25 it appears that the patterns and degrees ofAlz-50 immunoreactivity in neuronal perikarya and neu-rites are not diagnostic. Rather, these findings help un-derscore the notion that the filamentous inclusions ob-served in a number of neurodegenerative diseases arefundamentally similar.47-1 Moreover, the consistentlyhigher levels of Alz-50 immunoreactivity in AD + DNcompared with AD and AD + PD suggests that the un-derlying disease process responsible for AFI neuronaland neuritic degeneration is more severe and extensivein the setting of Down's syndrome. The presence of scat-tered AFI neurites and neuronal perikarya in controlbrains implies that some aspects of AD are features ofnormal aging.

The ALZ-EIA values better discriminated among thegroups than did the extent of immunohistochemical stain-ing: AD and AD + DN were distinguished from control bytheir higher ALZ-EIA levels in PBS extracts of cerebraltissue. The ALZ-EIA levels were correlated with the den-sity of AFI neurites, but not with the density of AFI neuro-nal cell bodies. Moreover, AD neurofibrillary tangles andneurites were better correlated with ALZ-EIA levels thanthey were with the densities of AFI neurons and neurites.Therefore, it appears that theAD pathologic process maybe better reflected by the levels of PBS-extracted Alz-50immunoreactivity than by the pattern of Alz-50 immuno-histochemical staining. At the same time, the findings


suggest that the levels of PBS-extractable immunoreac-tive protein only partly reflected the extent of Alz-50 im-munostaining, perhaps because some of the Alz-50 im-munoreactivity was associated with insoluble filamentousmatenal and therefore was not completely extracted inPBS.

In general, AD was readily distinguished from PD bythe significantly higher ALZ-EIA levels. Like the patternsof Alz-50 immunostaining, however, the ALZ-EIA levelsdid not consistently and unambiguously discriminateamong PD, AD, and AD + PD, particularly with respectto the parietal cortex and the amygdala. If one were touse 0.1 absorbance units per milligram of protein as thethreshold for diagnosing AD, then with rare exceptions,all cases of PD would be excluded, but many cases ofAD also would be missed, depending on the region ex-amined. The implication is that one could use the ALZ-EIA to distinguish between AD and PD, provided the tis-sue was obtained from the frontal or temporal lobe neo-cortex. This contrasts with the nearly identicaldistributions and densities of AFI neurites observed in ADand PD by immunohistochemical staining.

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disease-associated alz-50 immunoreactivity quantified in

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