Evidence that transmitter-containing dystrophic neurites precede those containing paired helical filaments within senile plaques in the entorhinal cortex of nondemented elderly and Alzheimer's disease patients

Brain Research, 619 (1993) 55-68 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

55

BRES 19080

Evidence that transmitter-containing dystrophic neurites precede those containing paired helical filaments within senile plaques in the entorhinal cortex of nondemented elderly and Alzheimer's disease patients William C. Benzing

a,c,,,

Daniel R. Brady h, Elliott J. Mufson c, and David M. Armstrong

a

a FIDIA-Georgetown Institute for the Neurosciences, Georgetown University, Washington, DC 20007 (USA) b Laboratory of Neuroscience, NIA/NIH, Bethesda, MD 20892 (USA) and c Department of Neurology, Rush-St. Luke-Presbyterian Medical Center, Chicago, IL 60612 (USA) (Accepted 2 March 1993)

Key words." Alz-50; Amyloid/3-protein; Immunocytochemistry;Neuropeptide; Paired-Helical Filament; Pathogenesis; Senile plaque

Within the amygdala of elderly subjects and patients with Alzheimer's disease (AD), we recently found evidence suggesting amyloid ~l-protein (A/3P) deposition occurs before the appearance of dystrophic neurites. Moreover, these data suggested dystrophic neurites initially lack evidence of cytoskeletal pathology although with time and further maturation, the dystrophic neurites display an altered cytoskeleton as evidenced by their immunoreactivity to Alz-50 and paired-helical filaments (PHF). These findings are of particular relevance to our understanding of the sequence of pathologic events in AD and thus it has become important to determine whether these events are unique to the amygdala or are representative of a more general pattern which can be found throughout the brain. Using a battery of antibodies to markers that are characteristic of AD pathology (i.e., A/3P, PHF, and Alz-50), three peptidergic neurotransmitters (neurotensin, somatostatin, and substance P), and one neurotransmitter biosynthetic enzyme (choline acetyltransferase), we examined the entorhinal cortex (EC) of three groups of subjects (AD, normal elderly, and a group of nondemented elderly with numerous senile plaques). The EC was studied, in part, because it is well recognized as a brain region displaying severe and, most importantly, early pathologic changes. Like the amygdala, we found evidence that amyloid /3-protein immunoreactive (A/3P-IR) and thioflavine-S-positive senile plaques occur within the EC prior to the appearance of transmitter-, Alz-50-, or PHF-immunoreactive dystrophic neurites. We also observed transmitter-immunoreactive dystrophic neurites in the absence of AIz-50 or PHF-immunolabeled dystrophic neurites and transmitter- and AIz-50-IR dystrophic neurites in the absence of those containing PHF. Collectively,these findings were similar to those seen within the amygdala and thus reinforced the concept that AflP deposition is the primary event in plaque pathology, and this deposition is subsequently followed by the appearance of dystrophic neurites which retain their transmitter phenotype yet lack an altered cytoskeleton. With time, these dystrophic neurites develop cytoskeletal alterations and become immunoreactive to Alz-50 and PHF.

INTRODUCTION

as a m e a n s of viewing early A D pathologic changes 1'5'6'24'34'44'45'55'57'59'68.I n this regard, we have exam-

Senile p l a q u e s in A l z h e i m e r ' s disease ( A D ) are primarily c o m p o s e d o f / 3 - a m y l o i d , dystrophic n e u r i t e s a n d glial e l e m e n t s 27'61. O n e a p p r o a c h to u n d e r s t a n d i n g the

i n e d the b r a i n s from a g r o u p of elderly p a t i e n t s who had n o clinical history of d e m e n t i a b u t u p o n n e u ropathologic e x a m i n a t i o n were f o u n d to have sufficient n u m b e r s of senile p l a q u e s to m e e t the K h a c h a t u r i a n

p a t h o g e n e s i s of senile p l a q u e s is to a t t e m p t to determ i n e the t e m p o r a l s e q u e n c e by which these various n e u r o n a l a n d n o n - n e u r o n a l e l e m e n t s come t o g e t h e r to form the plaque. T o assist these studies, investigators have b e g u n to e x a m i n e p a t i e n t s with D o w n ' s s y n d r o m e as well as n o n d e m e n t e d elderly with n u m e r o u s p l a q u e s

(1985) diagnostic criteria for A D . W e have previously called these individuals " h i g h p l a q u e n o n d e m e n t e d " ( H P N D ) cases 9,10. Recently, we d e m o n s t r a t e d that in selected regions of the amygdala in pathologically mildly affected H P N D

Correspondence: D.M. Armstrong, FGIN, Georgetown University, 3900 Reservoir Rd., Washington, DC 20007, USA. Fax: (1) (202) 687 1782. * Present address: Department of Neurology, Rush-St. Luke-Presbyterian Medical Center, 2242 W. Harrison St., Suite 200 Chicago, IL 60612, USA.

56 and AD cases, amyloid /3-protein (A/3P) deposition occurs alone in the absence of any appreciable neuritic pathology ~'9. In more severely affected cases, A/3P deposition continued to prevail but this time was observed together with dystrophic neurites that were immunolabeled by antibodies to the various transmitters, but not by antisera to Alz-50 or paired-helical filaments (PHF). Only as accumulations of A/3P increased further, as observed in the most severely affected cases, could dystrophic neurites be observed in which cytoskeletal changes were also apparent. In the present paper, we sought to determine whether this sequence of plaque formation was unique to the amygdala or was common to other brain regions. To answer this question, we chose to examine the entorhinal cortex (EC) because it, like the amygdala, exhibits AD pathology very early in the disease process and is densely innervated by many of the same neurotransmitter substances that were examined in the amygdala (i.e., acetylcholine, substance P, neurotensin, and somatostatin). However, the EC also is densely populated with neurofibrillary tangles thus providing the opportunity to examine the relationship between the formation of plaques and tangles. Furthermore, the pathologic changes in the EC may play an important a n d / o r primary role in the clinical expression of A D 13'31'36'64. Knowledge of the basic principles govern-

ing plaque formation is essential to our understanding of the pathogenesis of senile plaques. Moreover, an understanding of the primary events leading to the development of mature plaques will greatly assist our efforts in developing rational therapies aimed at halting a n d / o r slowing these pathologic lesions. MATERIALS AND METHODS Subjects Postmortem brain tissue was examined from: eight AD patients with both a clinical history and post-mortem neuropathologic verification of the disease; eight nondemented subjects with no clinical history of AD who exhibited sufficient numbers of senile plaques to meet the neuropathologic criteria for AD (Khachaturian 37) and thus were designated as H P N D cases; and five nondemented aged cases with little if any AD pathology which were classified as age-matched normal controls (NC). An additional younger NC (case #1, Table 1) was also examined and was used primarily to assess age-specific variation in immunostaining. Neuropathologic diagnosis of all cases was performed by the resident neuropathologist and was based, in part, upon examination of hematoxylin and eosin, thioflavine-S, and Bielschowsky silver stained tissue sections. Clinical evaluations of the nondemented cases were based largely upon retrospective analysis of medical records. Furthermore, interviews with the physicians and immediate family members of the H P N D cases provided additional evidence that these cases had not displayed any evidence of frank memory impairment or dementia. However, in the absence of neuropsychological testing it cannot be ruled out that these patients were exhibiting memory impairments which went undetected by family members or by their personal physician. The mean age, post-mortem interval (PMI), and brain weight of the cases were 80.2 years, 4.34 h, and 1,111 g for the AD patients;

TABLE 1

Case Information Table showing age (years), sex, post-mortem interval (PMI, h), brain weight (g), disease duration (years), and cause of death of the patients that comprised the three case groups in the entorhinal cortex study - normal controls (NC), high plaque nondemented (HPND), and Alzheimer's disease (AD) Cases are ordered by the rank ordering procedure described in the text. N / A , not available; ( - ) , not applicable.

Case # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

NC NC NC NC NC NC HPND HPND HPND HPND HPND HPND HPND HPND AD AD AD AD AD AD AD AD

Age

Sex

PMI

Brain weight

43 76 67 73 85 68 81 75 64 81 85 86 90 63 79 79 81 82 84 83 73 81

F M M M M M M M M M M M M F F M F M F F F M

6.00 3.50 21.00 5.00 18.00 6.50 4.50 6.00 5.75 7.00 6.00 3.00 7.00 5.00 4.75 7.00 5.00 3.00 3.50 6.00 3.00 2.50

N/A 1500 1400 1250 1 600 1.400 1 315 1 440 1210 1400 1320 1290 1250 1400 1 120 984 1 092 1210 1 100 1 142 1 070 1 170

Disease duration 3.00 10.50 6.00 3.00 9.00 6.00 2.50 16.00

Cause of death Cancer Heart failure Cancer Heart failure Pneumonia Cancer Liver cancer Respiratory arrest Hepatic/septic infection Pneumonia Prostate cancer Septic shock Respiratory failure Septic infection Respiratory failure Renal failure N/A Heart failure Pneumonia Respiratory failure Cardio-pulmonary arrest Heart failure

TABLE II

AD

HPND

Control

ENTO H

4 49 50 45 63 64 39 90

50.5 8.7

X S.E.M.

24.6 6.5

X S.E.M.

15 16 17 18 19 20 21 22

9 19 5 31 15 17 60 41

5.8 2.3

X S.E.M.

7 8 9 10 11 12 13 14

0 4 8 15 8 0

5.8 1.3

4 N/A 5 5 N/A N/A 4 11

6.6 1.2

2 3 10 9 8 9 9 3

2.8 0.8

0 3 2 2 5 5

42.9 7.5

26 26 45 25 24 80 57 60

41.9 10.9

90 20 22 31 93 23 25 31

4.3 4.1

0 0 0 0 1 25

12.4 1.4

13 14 10 6 11 10 17 18

11.4 1.6

14 16 10 9 10 6 7 19

0 0

0 0 0 0 0 0

THIO

E N T O III

PHF A L Z 5 0 [3-AMY neurons neurons

1 2 3 4 5 6

Case #

8.4 1.5

3 6 6 8 5 13 16 10

2.5 1.1

0 0 2 1 4 3 1 9

0 0

0 0 0 0 0 0

PHF

5.0 1.3

8 N/A 4 3 N/A N/A 8 2

4.2 0.8

2 4 2 7 6 9 3 2

0 0

0 0 0 0 0 0

ALZ50

1.9 0.4

3 0 1 3 2 2 3 1

1.6 0.4

1 0 1 2 2 4 1 2

0 0

0 0 0 0 0 0

1.1 0.3

1 2 1 2 1 0 2 0

1.0 0.2

0 1 1 1 1 2 1 1

0 0

0 0 0 0 0 0

SubP S O M

-

-

6 1 1 4 2.5 0.8

0 -

1.3 0.5

1 0 2 3 2 0 -

0 0

0 0 0 0 0

ChAT

1 2

1.8 0.7

0 2 0 5 2 4 0 1

0 0

0 0 0 0 0 0

NT

24.8 3.7

0 27 28 32 27 30 31 23

8.3 3.5

0 3 3 3 8 13 7 31

3.5 2.1

0 0 2 6 13 0

3.4 1.4

2 N/A 2 0 N/A N/A 5 8

9.4 2.9

4 10 6 10 8 4 4 29

2.2 0.4

1 2 2 2 4 2

PHF ALZ50 neorons neurons

22.3 1.9

27 30 17 23 23 18 26 15

16.1 2.9

30 11 22 7 7 15 14 23

1.8 1.6

0 0 0 0 0 11

[3-AMY

ENTO V

9.5 1.1

11 8 5 9 8 10 16 9

7.0 1.8

11 1 6 7 3 6 5 17

0.3 0.3

0 0 0 0 0 2

THIO

6.1 1.5

0 8 3 8 2 12 11 5

3.1 1.6

0 0 2 1 2 6 1 13

0 0

0 0 0 0 0 0

3.4 1.2

3 N/A 0 6 N/A N/A 6 2

4.3 0.9

1 6 2 6 2 8 6 3

0 0

0 0 0 0 0 0

PHF ALZ50

1.8 0.3

3 1 1 1 1 2 3 2

2.4 0.4

2 1 3 3 2 1 4 3

0.2 0.2

0 0 0 0 0 1

0.5 0.3

2 0 1 1 0 0 0 0

1.4 0.4

0 0 2 2 1 1 3 2

0.2 0.2

0 0 0 0 0 1

SubP S O M

1.8 0.3

2 2 2 1

3 1

1.4 0.8

0 0 0 1 0 2 7 1

0 0

0 0 0 0 0 0

NT

-

-

1 -

0.5 0.2

0 0 0 1 1 1 -

0 0

0 0 0 0 0

ChAT

52.4 11.5

0 30 15 81 63 80 73 77

23.7 13.5

0 1 3 7 12 17 36 114

2.8 1.4

0 1 0 8 6 2

5.2 1.1

1 N/A 6 7 N/A N/A 7 5

9.9 1.9

3 13 6 11 9 9 7 21

2.5 1.2

0 3 0 1 8 2

PHF ALZ50 neurons neurons

Table showing the mean density per 200xoptieal field, within Layers II (ENTO II), III (ENTO III), and V (ENTO V) of the entorhinal cortex, of A/3P-, PHF, Alz-50-, substance P- (Sub P), somatostatin- (SOM), neurotensin- (NT), or choline acetyltransferase- (CHAT) immunoreactive plaques, thioflavine-S-stained plaques and PHF- and Alz-50-containing neurons each of the normal control (NC), high plaque nondemented (HPND), and Alzheimer's disease (AD) cases. Within each group, the cases are listed in the order of pathologic severity, as describedin the text. X, mean; S.E.M., standard error of the mean; ( - ), insufficient staining density to evaluate; N / A , tissue not available for staining.

Mean number o f plaques or neurons per 200 × optical field in layers II, III, and V o f entorhinal cortex

"--..I

58 78.1 years, 5.5 h, and 1.328 g tor the ttPND: and 73.8 years, 10.0 h, and 1,430 g for the five age-matched NC cases. AD patients had an average disease duration of 7.0 years (range - 2.5-16 years). None of the subjects had other confounding neurological or neuropathological disorders.

Tissue preparation Brain tissue was processed according to previously described procedures It'15'53. Briefly, blocks of tissue containing the amygdala, entorhinal cortex, uncus of the hippocampus and surrounding temporal cortex were cut in the coronal plane and placed in 0.1 M phosphate buffer (PB, pH = 7.4) containing 4% paraformaldehyde for 24-48 h at 4°C and then cryoprotected in graded sucrose concentrations (10-30%) in PB for several days. The tissue was then sectioned at 40/xm on a sliding, freezing microtome, collected in a 1 in 18 series, and stored in an ethylene glycol/glycerol/phosphate buffer cryoprotectant solution. Selected tissue sections, adjacent to those used for immunocytochemistry, were stained for Nissl substance in order to determine the various layers of the entorhinal cortex.

lmmunohistochemistry Antisera. Tissue sections were immunolabeled with antibodies to the following Alzheimer-related antigens: (1) amyloid /3-protein 35 ('R1280' from Dr. D. Selkoe), (2) PHF (by two different antisera, 'Pare '32 provided by D. Selkoe and 'PHFI '2s provided by Dr. P. Davies), and (3) AIz-5067 (Abbott Labs). The following antisera against 3 neuropeptides and one transmitter biosynthetic enzyme were used: neurotensin 11 (NT), somatostatin7 (SOM), substance plg, and choline acetyltransferaseTM (CHAT) (provided by L. Jennes, R. Benoit, Accurate Chemicals and L.B. Hersh, respectively). Single-immunolabeling. Free-floating tissue sections were processed for immunocytochemistry using the ABC method of Hsu and Raine (1981) as described previously 3'I1. Briefly, tissue sections were incubated in primary antiserum diluted in Tris-buffered saline (TBS) with 1% bovine serum albumin (BSA) and 0.25% Triton X-100 (Sigma) as follows: 'R1280' (1:6000), 'PAM' (1:4000), PHF1 (1:60,000), Alz-50 (1:300), substance P (1:500), NT (1:6000), SOM (1:4000) or ChAT (1:1800). Tissue then was incubated in one of the following biotinylated secondaries diluted 1:200 in TBS with 1% BSA: goat anti-rabbit (Vector Labs) for 'R1280', 'Pam', NT, SOM and CHAT; rabbit anti-rat (Vector Labs) for substance P; and goat anti-mouse (Jackson Immunoreagents) for 'PHFI' and Alz-50. After incubation in avidin-biotin-peroxidase complex (Vector, ABC Elite) diluted 1 : 100 in TBS, the antigens were visualized by reacting with /I.05% diaminobenzidine (DAB) and 0.04% NiCI in Tris-hydrochloride buffer. Sections were mounted on chrome-alum coated glass slides, counterstained with 1% thioflavine-S and coverslipped using Entellan (EM Science). As a control for nonspecific staining, sections were either incubated in the initial incubation media minus the primary antibody or preabsorbed with microgram quantities of synthetic antigen (peptidergic antisera only) and otherwise processed normally. In addition to the avidin/biotin immunolabeling procedure, Alz50 IgM was visualized using an indirect peroxidase procedure 15'67. In this procedure, tissue sections were incubated in Alz-50 antiserum diluted 1:100 in phosphate buffer containing 5% nonfat dry milk (Carnation) followed by incubation in goat anti-mouse peroxidase (Sigma), and visualized using DAB. Double-immunolabeling. From each case, three adjacent sections were dual immunostained for either substance P and AflP (R1280), PHF (PHF1) and A/3P (R1280), or substance P and PHF (PAM) using two methods of visualization. The first technique utilized DAB and amino-ethylcarbazole (AEC) as the chromogens and yielded homogeneous brown/black and red reaction product colors, respectively. Alternatively, the second method substituted benzidine dihydrochloride (BDHC) for DAB. The BDHC yielded a highly granular blue/green reaction product which clearly distinguished co-localized antigens when superimposed on the red homogeneous AEC label. Collectively, these studies allowed us to assess whether dystrophic neurites and A/3P-immunoreactive (A/3P-IR) plaques were co-dis-

tributed and whether substance P and PHF were co-localized within individual dystrophic neurites.

Quantitatit~e morphornet~' Dystrophic neurites were defined as neuritic processes which were grossly swollen in appearance. The extent to which these neurites were enlarged varied considerably and ranged from individual swollen varicosities to large 'grape-like' clusters of immunoreactivity (see Armstrong et al., 1989). Dystrophic neurites were distinct from neuropil threads which were seen as slightly thickened irregularly shaped neuritic processes randomly dispersed within the neuropi114. Neuropil threads showed no predilection for A/3P deposition, whereas, dystorphic neurites were almost exclusively found associated with A/3P. A clustering of two or more dystrophic neurites was operationally defied as a plaque. Thioflavine-S-stained plaques and plaques immunoreactive for each of the eight antibodies (i.e., R1280, PAM, PHF1, AIz-50, substance P, SOM, NT & CHAT), were counted using a manual push button counter. Neurons immunolabeled by either PHF or Alz-50 also were counted. For each case, we obtained plaque counts from two single-immunolabeled sections per antigen. Within each tissue section, we counted at least 4 different 200x fields from within layer II, III, and V of the EC. Counts were limited to a region located between the intrarhinal sulcus and the collateral sulcus. All 200x optical fields were randomly selected within each section but were located within approximately the same coronal levels of the EC. If the variance was high among the four sampled 200x fields, additional 200 x fields were counted up to a maximum number of eight. From each case, the counts within each of the three EC lamina were then averaged with those obtained from the corresponding layer in the second tissue section. Thus, the final tabulated value represents the data from a minimum of eight optical fields. In addition, for each case, the counts within the three lamina in the EC were averaged together to provide a single mean density value for each marker per subject. To facilitate analysis of the temporal order of pathologic changes, the cases were ordered by increasing pathologic severity. To accomplish this, the cases were rank ordered according to the density of thioflavine-S-stained, AflP-, PHF-, and Alz-50-immunoreactive plaques as well as the density of PHF- and Alz-50-immunoreactive neurons. Next, for each case, the ranking it received from each of the six individual rank orderings was then summed. This sum, in turn, was then used as the basis for a final rank ordering of pathologic severity (as seen in Tables I and II).

Statistical analysis Within each of the three EC lamina and on the mean data across the EC, one-way analyses of variance (1×3 ANOVAs) and associated post-hoc t-tests (Tukey's) were performed on the mean number of plaques or neurons per 200 x field (i.e., density) for each marker. Pearson product-moment correlational analyses also were performed and included the subject's age, post-mortem interval, brain weight, and disease duration (disease duration in correlations involving AD cases only). Separate correlational analyses were performed that included: (i) all subjects, (ii) HPND and AD only, and (iii) AD only.

RESULTS In the

present

study, we examined

Nissl s t a i n e d

t i s s u e s e c t i o n s a n d d i v i d e d t h e E C i n t o a six l a y e r e d s t r u c t u r e as d e s c r i b e d by A m a r a l a n d I n s a u s t i (1990) in their review of the cytoarchitecture of the human EC. The nomenclature for the various lamina of the human EC,

however, varies depending

e x a m p l e , o u r l a y e r II is t e r m e d

on

the

author.

For

P r e - a by Br aak a n d

B r a a k (1985), w h i l e l a y e r III is t h e s a m e as t h e i r Pre-/3 a n d Pre-y. Similarly, layer VI c o r r e s p o n d s

to B r a a k

59 and Braak's layer Pri-y, while layer V corresponds to Pri-a and possibly part of Pri-/3. In addition, our layer V corresponds to layer IV and possibly layer V of Van Hoesen et al. (1991) and H y m a n et al. (1984). Finally, layer IV corresponds to the lamina dessecans 2. Plaques were consistently and predominantly observed within layers III and V (Fig. 1). In contrast, the density of plaques was often variable in the remaining lamina, however, in general they were relatively few in number. For this reason, quantitative analysis of plaque

density was restricted to layers III and V. Similarly, layers II, III and V exhibited the highest propensity for P H F and Alz-50 immunostained neurons, thus quantification of the density of these immunoreactive structures was restricted to these three lamina.

Thioflavine-S Thioflavine-S stained numerous plaques in the EC of all A D and H P N D cases but rarely in the NC (Table II). In fact, thioflavine-positive plaques were seen in

Fig. 1. Laminar distribution of PHF-IR (B,D,F) and AflP-IR (A,C,E) within the entorhinal cortex of a pathologically'mild' (A&B, case #8) and 'severe' (C&D, case #12) HPND case and in a pathologically 'severe' (E&F, case #21) AD case. Arrows in (D) and (F) indicate examples of PHF-IR neuritic plaques. Note in (C) the subpial A/3P deposition within layer I of the more severelyaffected HPND case. Bar = 0.5 mm.

60 only one NC case (#6). Fluorescent plaques were found mostly in layers III and V, although a few plaques were occasionally seen in layers II and VI. Thioflavine-S revealed classical, primitive and diffuse type plaques. Classical plaques were most prevalent in layer III in both AD and HPND cases although numerous primitive and diffuse plaques were also located within this layer. Layer V consisted mainly of primitive and diffuse plaques although some classical plaques were present. AD and HPND cases exhibited similar densities of thioflavine-stained plaques in both layers III and V and did not differ from each other statistically (Fig. 2 and Table II, III and IV). A [3P-Immunoreactivity Antibodies to A/3P immunolabeled numerous plaque-like deposits of extracellular granular material in HPND and AD cases but rarely in the NC. A/3P-immunoreactive (A/3P-IR) plaques were seen in all lamina but were primarily observed in layers III and V. Several cases exhibited a subpial 'ribbon' of A/3P-immunoreactivity in layer I (Fig. 1C), similar to that described previously 65'69. Layer III generally exhibited more plaques than layer V, particularly in case #'s 7, 11, 13, 20 and 22 (Table II and Fig. 2). In addition,

antisera to A/3P revealed, on average, nearly two to three times more plaques than thioflavine-S. The NC cases did not exhibit any A/3P-IR plaques, except for cases #5 and #6. Case #5 contained a small number of A/3P-IR plaques in layer III, while case #6 exhibited a small number of plaques in layer V with more dense accumulations in layer III (Table II). In contrast to NC cases, HPND and AD cases exhibited high densities of A/JP-IR deposits in layers III and V and differed significantly from NC (Tables II and IV). AD and HPND cases, however, did not statistically differ from each other in the density of these deposits within either layers III or V or when averaged across the EC (Fig. 2, Tables II-IV). PHF and Ah-50 The antisera to PHF and Alz-50 immunostained dystrophic neurites, neuropil threads, and neuronal perikarya. The PHF antisera also immunostained many extracellular 'ghost' tangles 33. The staining properties of the two antisera against PHF (i.e., PAM and PHF1) were nearly identical and thus the findings obtained from them were pooled together. NC cases did not exhibit any PHF-immunoreactive (PHF-IR) dystrophic neurites. However, within nearly

T A B L E 1II

Analysis o f meaned data across all 3 lamina o f the E C T a b l e showing (1) the m e a n density of p l a q u e s or P H F or Alz-50 n e u r o n s w h e n a v e r a g e d across the e n t o r h i n a l cortex and (2) the results of the statistical analysis of these values across the t h r e e case g r o u p s showing the F - r a t i o a n d the possible c o m b i n a t i o n s of t-test values

Means

A/3P-IR deposits Thioflavine-S p l a q u e s PHF-IR plaques AIz-50-IR p l a q u e s Sub P - I R p l a q u e s SOM-IR plaques NT-IR plaques PHF-IR neurons AIz-50-1R n e u r o n s

Statistics A/3P-IR deposits Thioflavine-S p l a q u e s PHF-IR plaques AIz-50-1R p l a q u e s Sub P - I R p l a q u e s SOM-IR plaques NT-IR plaques ChAT-IR plaques PHF-IR neurons AIz-50-IR n e u r o n s

Case groups NC Mean (S.E.M.)

HPND Mean (S.E.M.)

AD Mean (S.E.M.)

3.08 0.17 0 0 0.17 0.17 0 4.06 2.50

29.00 9.19 2.81 4.31 2.00 1.19 1.56 18.96 8.63

32.63 10.94 7.25 4.20 1.81 0.81 1.63 42.54 4.80

(2.98) (0.17)

(0.11) (0.11) (1.73) (0.72)

(5.87) (1.46) (1.29) (0.84) (0.25) (0.23) (0.49) (7.07) (1.48)

(3.25) (1.08) (1.48) (0.78) (0.31) (0.21) (0.46) (7.01) (0.74)

ANOVA

Post hoc t t;alue

F2,19

N C vs. HPND

N C vs. A D

HPND vs. A D

3.96 5.35 1.55 4.17 4.85 3.43 2.49 2.71 1.60 3.64

4.52 6.38 4.00 3.64 4.36 2.17 2.59 0.25 4.14 1.22

0.60 1.12 2.64 0.10 0.54 1.36 0.11 2.66 2.74 2.15

11.62 22.51 8.36 10.19 13.63 5.91 4.12 4.94 8.98 6.88

*** *** ** * *6 *** ** * * ** * *6

* P < 0.05; * * P < 0.01; * * * P < 0.001; 6, F2.16.

** ** ** ** ** * * **

** ** ** ** ** * * **

*

* * *

61 all H P N D and AD cases, plaque-like aggregates of P H F - I R dystrophic neurites were observed and these were found mainly in layers III and V (Table II). A few P H F - I R plaques also were seen in layers II and VI. AD cases exhibited three times more P H F - I R plaques in layer III compared to H P N D cases and two times more P H F - I R plaques in layer V. The higher density of P H F - I R plaques in AD compared to H P N D occurred despite similar densities of A/3P-IR deposits or of thioflavine-positive plaques in these same two lamina. While AD cases exhibited significantly more PHF-IR plaques than H P N D in layer III, no statistical difference was seen in layer V between the two case groups (Fig. 2 and Table IV). The lack of statistical significance within layer V can be attributed to the variance induced by one of the H P N D cases (#14) that was characterized by a very high density of PHF-containing plaques. Without this case in the analysis, the density of PHF-containing plaques differed significantly between the AD and H P N D cases (t = 2.57; P < 0.05) in layer V as well. Interestingly, while the AD cases contained significantly higher densities of P H F - I R plaques than NC cases in both laminas III and V, the H P N D cases did not differ statistically from the NC (Fig. 2 and Table IV).

In contrast to P H F - I R plaques, the density of Alz50-positive plaques in layers I I I & V was not significantly different between H P N D and AD cases (Tables II and IV; Fig. 2). NC cases did not exhibit any Alz-50-immunoreactive dystrophic neurites. Although neuropil threads were often observed within plaques, they showed no particular predilection for plaques and could be found scattered in the neuropil in a near random-like fashion. PHF- and Alz-50immunoreactive neuropil threads were particularly prevalent in areas that contained numerous P H F or Alz-50 containing neurons such as layers II and V of the EC and to a lesser extent, layer III. Neuropil threads appeared to be associated most often with tangle bearing neurons and were most dense in their immediate vicinity.

Transmitter-containing plaques The antibodies against the four transmitters revealed beaded fibers, dot-like puncta, and dystrophic neurites. In addition, a small number of immunosrained neurons were revealed by antisera to somatostatin, substance P, and in some of the cases, neurotensin. Transmitter-immunoreactive dystrophic neurites were most frequently observed within plaques in

TABLE IV

Statistical analysis o f data within individual E C lamina T a b l e s h o w i n g the results f r o m the statistical analysis o f the m e a n p l a q u e a n d P H F a n d Alz-50 n e u r o n c o u n t s ( s e e n in T a b l e I I ) within e a c h of the three lamina

ANOVA

Post hoc t value

F2,19

N C vs. H P N D

N C vs. A D

HPND vs. A D

1.84 2.52 *

4.38 1.76

2.74 0.65

2.95 5.76 1.44 3.67 2.66 3.08 1.95 1.01 2.32

** **

3.12 6.27 4.83 4.06 3.12 3.46 2.60 4.31 0.35

** ** ** ** ** ** * **

0.28 0.55 3.66 0.66 0.49 0.42 0.84 3.56 1.82

4.23 3.42 1.54 3.80 3.92 2.31 1.61 1.27 3.30

** **

5.59 4.70 3.02 2.93 2.62 0.37 2.00 3.01 1.08

** ** ** ** *

1.60 1.39 1.60 0.67 1.41 2.10 0.54 1.88 1.98

Layer H PHF-IR neurons AIz-50-IR neurons

9.93 * * * 3.29 6

Layer III A / 3 P - I R deposits Thioflavine-S plaques PHF-IR plaques Alz-50-IR plaques Sub P - I R p l a q u e s SOM-IR plaques NT-IR plaques PHF-IR neurons Alz-50-IR neurons

5.95 23.19 12.95 9.81 5.43 6.89 3.61 10.85 3.15

** *** *** * *6 * ** * *** 6

** * **

*

Layer V A f l P - I R deposits Thioflavine-S plaques PHF-IR plaques AIz-50-IR plaques Sub P - I R p l a q u e s SOM-IR plaques NT-IR plaques PHF-IR neurons Alz-50-1R n e u r o n s * P <0.05; ** P<0.01;

16.57 * * * 11.46 * * * 4.59 * 7.77 * *6 7.80 * * 3.35 * 2.21 4.67 * 5.71,8 * * * P < 0.001; 8,

F2,16,

** ** *

**

*

~ / ~

A

Layer IIi

/llNc NHPND ~aD

5O

.

.

.

.

40

x

%

xo

| ~.

o

::

e

cases (Fig. 2 and Table IV). These latter observations can be attributed to a paucity of SOM-immunolabeled fibers within layer V of the EC in AD cases. The density of ChAT-containing plaques was not evaluated statistically within the EC of AD cases because CHATimmunoreactivity was extremely depleted in the EC. However, ChAT-IR dystrophic neurites were seen in the EC of the H P N D cases and occurred predominantly within layer III.

z I m m u n o l a b e l e d neurons

g o ~

I

[ B

Layer V

I

~o ~o

< Fig. 2. Graphic representation of the mean density, per case group, of immunolabeled plaques as seen in Table II within layers Ill (A) and V (B) of the EC. Scale bar on the y-axis represents mean density of immunoreactive plaques per 200 × field. Probability of statistically significant differences: ** P < 0.01, HPND and AD cases are both significantly different from NC cases; ~ P < 0.05, ~ P < 0.01, HPND cases are significantly different from AD cases; § P < 0.05, '~§ P < 0.01, HPND cases are significantly different from NC cases; o p < 0.05, oo p < 0.01, AD cases are significantly different from NC cases.

layer III although many were observed within lamina V, and to a lesser extent, within layers II and VI (Table II). Importantly, the density of transmitter-immunoreactive dystrophic neurites did not follow the normal innervation pattern or density of any of the transmitters but rather paralleled the density of A/3P deposits. It is of interest that the only NC case (#6) to exhibit transmitter-containing dystrophic neurites also displayed somewhat high densities of A/JP-IR plaques although few thioflavine-stained plaques were observed (Table II). All the H P N D and AD cases, however, exhibited transmitter-containing dystrophic neurites in plaques. Compared to each other, H P N D and AD cases exhibited similar densities of somatostatin-, substance P- and neurotensin-containing plaques in layer III, and of substance P- and NT-containing plaques in layer V. However, significantly more SOM-containing plaques were seen in layer V in H P N D than in AD

PHF-positive neurons were observed within all lamina of H P N D and AD cases except layers I and IV and were most dense within layers II and V. Quantitatively, the mean density of PHF-IR neurons in layer II did not differ from layer V in either the A D or H P N D cases (Table II and Fig. 3). Qualitatively, however, the immunoreactive neurons in these two lamina differed greatly. For example, in layer II, the majority of the PHF-IR profiles appeared as extracellular tangles whereas in layer V, most PHF-IR neurons were observed to lie within discernable neuronal perikarya with relatively few appearing extracellular. PHF-IR neurons were also observed within layer III, and to a lesser extent, in layer VI. PHF-IR neurons were seen in all of the NC cases, except the single non-elderly case (#1) (Table II). The majority of these neurons occurred primarily in layer II, although in four of the six NC cases immunolabeled neurons were observed also in layer V and three of the six exhibited a few PHF-IR neurons in layer III. In contrast, all HPND and AD cases displayed PHF-IR neurons in layers II, III and V except for one H P N D case (#7) and one AD case (#15) which did not display any PHF-IR neurons in laminas III and V. Compared to NC cases, the H P N D cases exhibited a greater density of PHF-IR neurons in each of the three lamina (Fig. 3). H P N D cases averaged nearly four, two and eight times the density of PHF-IR neurons in layers II, III, and V, respectively, when compared to NC. However, despite these differences, the H P N D cases, as a group, did not differ significantly from NC cases in the density of P H F neurons in layers III or V (Table IV). As with P H F - I R plaques, the lack of statistical significance in layer V between H P N D and NC cases, in this instance, could be attributed to the extremely large variance for the H P N D cases, again contributed by case #14. Removing case #14 from the analysis, the H P N D cases then exhibited a statistically higher number of PHF-IR neurons in layer V than the NC (t = 3.72; P < 0.01) although significance was still not achieved in layer III.

63 Differences in density of P H F - I R neurons were even more profound in the AD cases compared to NC cases (Fig. 3). For example, AD cases contained nearly seventeen times the number of P H F - I R neurons in layer V, ten times more in layer II and seven times more in layer III, than NC cases. Furthermore, AD cases contained nearly double the number of P H F - I R neurons in all three layers compared to H P N D cases. The AD cases exhibited statistically significant higher densities of P H F - I R neurons in all three lamina when compared to the H P N D and NC cases (Tables 3 and

4). Alz-50-immunoreactive (Alz-50-IR) neurons exhibited a laminar organization similar to P H F - I R neurons although they were much less abundant (Table II). Interestingly, all of the NC cases exhibited Ah-50 neurons, even the 43-year-old case (#1). Alz-50-IR

A

Z X

2 Z

ALZ50

I INc ~HPND ~AD

50 40 30 !

20

t§

o Layer II

LayerIll

LayerV

6O O "~

50

0 0

40

~

ao

0 Z

2o

neurons were most dense, however, in the H P N D cases. In fact, H P N D cases exhibited nearly four times the number of Ah-50-IR neurons in layers III and V compared to NC cases, and nearly three and two times the number of Alz-50-positive neurons in layers III and V, respectively, compared to AD cases (Fig. 3). Differences in the density of Alz-50 neurons in layer II were much less, however, between H P N D and AD. Despite these differences, the marked variance displayed by the H P N D and AD cases negated these data from being statistically different when each lamina was analyzed separately, however, when averaged across the EC, the number of Alz-50-IR neurons was significantly higher in H P N D than AD cases (Table III). Furthermore, H P N D cases also differed significantly from the NC in the density of Alz-50 neurons in each of the three lamina and when averaged across the EC (Tables 3 and 4).

Double-immunolabeling Serially adjacent tissue sections, double-immunolabeled for A/3P and PHF, A/3P and substance P, or PHF and substance P, confirmed many of the observations in the EC that were obtained from examination of the single-immunolabeled sections. Most importantly, PHF-IR and substance P-IR dystrophic neurites were not observed outside of A/3P-IR plaques in dualimmunostained tissue sections. These data were in contrast to those obtained in single-immunolabeled sections, counterstained with thioflavine-S. In these preparations, we observed numerous instances of dystrophic neurites alone in the neuropil without any apparent association with thioflavine-S positive senile plaques. Furthermore, dual-immunolabeling confirmed that P H F - I R neuropil threads exhibited little predilection for A/3P-IR plaques. Finally, dual immunostained tissue demonstrated that substance P and P H F were co-localized, in many instances, within the same dystrophic neurites although they were also observed in separate neurites.

Correlational analyses o Layer II

Layer III

Layer V

Fig. 3. Graphic representation of the mean density, per case group as seen in Table II, of Ah-50-IR (A) and PHF-IR (B) neurons within layers II, III, V of the entorhinal cortex. Scale bar on the y-axis represents the mean density of immunolabeled neurons per 200× optical field. ALZ-50, Alz-50-immunoreactiveneurons. Probabilityof statistically significant differences: s~P < 0.01, HPND cases are significantly different from AD cases; §§P < 0.01, HPND cases are significantly different from NC cases; OOp < 0.01, AD cases are significantlydifferent from NC cases.

The density of AD-specific markers (A/3P, P H F and Ah-50) failed to correlate with age, brain weight or post-mortem interval. Thioflavine-positive plaques, however, were negatively correlated with age (r = 0.63; P < 0.01) when the H P N D and AD cases were considered together (i.e., omitting the NC cases from the analysis). Statistically significant positive correlations were observed between the density of A f l P - I R plaques and the density of PHF-containing plaques, thioflavineS plaques and substance P-containing plaques (r's >_

64 0.72, P < 0.05). Thioflavine-S plaques, however, were positively correlated with PHF-IR neurons, PHF-IR plaques, and substance P-containing plaques (r's >_ 0.56; P < 0.01). PHF-IR neurons were positively correlated only with PHF-IR plaques and thioflavine-positive plaques (r's _> 0.86; P < 0.01). Finally, Alz-50-containing plaques were positively correlated with the density of each of the neurotransmitter-containing plaques (r's > 0.57; P < 0.05). When correlational analyses were performed using only the AD cases, the following two significant correlations occurred: PHF-containing plaques positively correlated with A/3P-IR plaques (r = 0.79; P < 0.05) and AIz-50-IR plaques correlated with substance Pcontaining plaques (r = 0.89; P < 0.01). Likewise, when only the eight H P N D cases were considered, statistically significant positive correlations existed between the density of PHF-IR plaques and PHF-IR neurons (r = 0.81, P < 0.05), between PHF-IR plaques and Alz-50-IR neurons (r = 0.83; P < 0.01), and between PHF-IR neurons and Alz-50-IR neurons (r = 0.80, P < 0.01). DISCUSSION The present study supports the findings from our previous investigation of the amygdala 8'9. In that study, we demonstrated A/3P likely precedes the formation of dystrophic neurites and that the earliest appearance of dystrophic neurites can be observed with antibodies directed against the native transmitter system(s) of the neurites but not with markers of cytoskeletal change. Evidence of neurofibrillary pathology in these regions of early pathologic changes occurs only as the overall degree of pathologic severity is increased. In brief, both studies suggest that A f l P is the primary pathologic event in plaque development followed by the formation of PHF-negative dystrophic neurites in plaques and later by those containing PHF. However, in contrast to the amygdala, the EC contained such a high density of Alz-50- and P H F - I R structures that it was more difficult to interpret the temporal order of pathologic events. In fact, in the present study, the earliest pathologic changes were seen only within the NC cases and a couple of the H P N D cases. These findings reinforced the concept that the pathology in the EC may precede that occurring in the amygdala 13'57. For example, in the EC, we observed only one case (#6) which exhibited transmitter-immunoreactive dystrophic neurites in plaques in the absence of Alz-50 or P H F in dystrophic neurites and only two cases (#5 and 6) which displayed plaques in a lamina in the absence of any dystrophic neurites. Likewise, only two H P N D

cases ( # 7 and 8) and one AD case (#15) exhibited dystrophic neurites immunostained with antibodies against Alz-50 or one of the various transmitters in the absence of PHF-IR dystrophic neurites. In the remaining HPND and AD cases, however, PHF-IR dystrophic neurites were seen within plaques along with Alz-50 or transmitter-immunolabeled neurites. In interpreting the data obtained from the EC and in the comparisons of these data with those of the amygdala, it is important to keep several factors in mind. First, the EC exhibited many more Alz-50 and PHF-IR neurons than were seen within the amygdala, thus it would be predicted that the EC would have a higher density of Alz-50 or PHF-containing plaques. This notion is reinforced by the fact that in the EC, the density of PHF-IR neurons significantly correlated with increasing PHF-IR plaques. Secondly, much evidence suggests that the entorhinal cortex may be among the earliest regions of the brain to develop senile plaques and tangles 13'2°'2~'42'5v. Certainly, our data would suggest an earlier involvement of the EC than the amygdala. Illustrating this point are the H P N D cases in which we observe a pathologic profile far exceeding that observed in the amygdala 8'9. For this reason, it became necessary to examine the NC cases in order to observe the earliest indications of pathologic change. Similar conclusions were reached by Price et al. 57 in their study of 25 aged demented and non-demented individuals and by Braak and Braak ~3 in their study of 83 cases of mixed etiology. From the observations in these NC and H P N D cases, it is apparent that the deposition of A/3P likely precedes neuritic alterations. This deposition, in turn, affects neurites in the immediate vicinity of the amyloid deposition in a manner that causes them to swell and to become aggregated into the periphery of the plaque. The neurites which participate early in plaque formation lack PHF. These data are in agreement with Hansen et al. (t991) who demonstrated that despite comparable numbers of neuritic plaques in the EC of cases with AD and of cases with Lewy Body disease, the neuritic plaques in the Lewy Body cases were PHF-negative. Furthermore, the PHF-negative neuritic plaques occurred despite the presence of PHF-IR neurons in the Lewy Body subjects. Similarly, it has been shown that GAP-43, a-l-antichymotrypsin, chromogranin A, spectrin, casein kinase II, synaptophysin and tau can be localized to dystrophic neurites in plaques prior to P H F 4'17'39'49-51'54'60. Furthermore, our dual-immunolabeling suggests that virtually all dystrophic neurites are associated with A/3P-IR plaques even when they are not associated with thioflavine-S positive plaques.

65 Although the swelling or distention of the neurites precede PHF formation, they may be closely associated with cytoskeletal alterations as indicated by the early appearance of Alz-50 within these swollen neurites 3s'4°'67. Importantly, in our study, the density of Alz-50-containing dystrophic neurites in plaques positively correlated with the density of transmitter-containing dystrophic neurites but not those containing PHF. These data suggest the initiation of cytoskeletal changes begin at a time during which the dystrophic neurite is still expressing the native transmitter. In support of these findings is the study of Lenders et al. 41 who studied the hippocampus of patients with AD by immunostaining 12 /zm cryostat sections with either substance P, somatostatin, cholecystokinin, or neuropeptide Y and then by use of an elution-restaining procedure, removed the stain and then immunostained again using antisera against tau/PHF. These authors first noted that the vast majority of transmitter-immunoreactive dystrophic neurites were located in fairly immature plaques suggesting that these types of dystrophic neurites are involved in the early stages of plaque formation. Secondly, dystrophic neurites that were initially immunostained for the peptidergic transmitters exhibited little tau/PHF-immunoreactivity. They also noted that the larger the cluster of peptidergic dystrophic neurites, the less likely it was to stain for tau/PHF. They concluded that neurites degenerate after first becoming 'big and ballooning' at which stage they still contain large amounts of their normal peptidergic secretion product. However, at this early stage, they are not immunolabeled by anti-tau/PHF. Degradation and t a u / P H F formation follow diminished peptidergic content. In our double-labeling study, we saw numerous instances of substance P and PHF co-localized within individual dystrophic neurites. The majority of dystrophic neurites, however, were immunolabeled by PHF only and were substance P-negative. The precise relationship between PHF formation and A/3P deposition is unknown, although some studies would suggest they are independent processes 56'58'65'66. For example, our observation that a small number of PHF and Alz-50 containing neurons exist in nearly all of the NC cases in the absence of A/3P deposition suggests that, at least within the perikarya, the formation of neurofibrillary pathology may occur independent of A/3P deposition. Our inability to show any significant correlation between the density of A/3P deposition and the density of Alz-50- or PHF-IR neurons further indicates a dichotomy in their formation. We did see, however, a significant positive correlation between the density of the A/3P and PHF-IR dystrophic neurites suggesting that the formation of PHF

in dystrophic neurites may be linked to A/3P deposition, which has been suggested by others 66. In contrast to PHF-IR dystrophic neurites, we saw elevated densities of neuropil threads in the vicinity of increased PHF- or Alz-50-IR neurons but not in association with plaques. Together, these data indicate a potential relationship between PHF-IR dystrophic neurites and plaques, while neuropil threads may be more related to tangle formation as suggested by Braak and Braak (1986). However, a significant positive correlation also existed between PHF-IR neurons and PHF-IR plaques somewhat clouding these conclusions. Several investigators have suggested A/3P may not be a sufficient and necessary condition for the dementia to occur in AD 22-24'26'43'47'52. These same authors suggest the dementia in AD may be more associated with neurofibrillary pathology rather than /3-amyloid accumulation. Crystal et al. (1988), in a study of demented and nondemented elderly, reported that of many pathologic measures, only the number of cortical neurofibrillary tangles could distinguish between demented and nondemented subjects, whereas plaque count could not. Likewise, in our study, differences in the density of PHF-IR plaques and neurons were the only markers that consistently distinguished our HPND cases from the AD cases. Even though all of our HPND cases exhibited varying degrees of PHF formation, the AD cases exhibited significantly higher densities of PHF-IR plaques and neurons. These statistically significant differences occurred despite similar densities of plaques revealed by AflP immunoreactivity or thioflavine-S. Interestingly, the HPND cases exhibited more Alz-50 containing neurons than AD cases, suggesting the HPND cases had more neurons that were potentially in an earlier stage of neurofibrillary change than the AD cases. When correlational analyses were performed only on the data from the HPND cases, Alz50-positive neurons correlated positively with PHF-IR neurons, further suggesting a link between the two populations of neurons. These data suggest that a continuum in the development of neurofibrillary pathology occurs, such that if the HPND had lived longer, the Alz-50-IR neurons likely would have developed into PHF-containing neurons. Whether an increase in PHF-IR neurons would result in a corresponding increase in the density of PHF-IR plaques is unclear. However, as mentioned previously, a positive correlation did occur between PHF-containing neurons and PHF-IR dystrophic neurites. With a further increase in neurofibrillary pathology, these individuals then may have begun to exhibit behavioral changes characteristic of dementia, if they had lived longer. In

66 this regard, Dayan 2°,21 reported that the single differentiating feature between clinically normal aged controls with high numbers of plaques and A D cases was the presence of significantly more tangles in the AD cases. Since the only other measure differentiating our H P N D from the A D cases, besides a loss of brain weight and increased neurofibrillary pathology, was the presence of frank dementia, our findings too suggest that P H F formation or factors that are related to tissue loss such as loss of neurons or fibers 46 may be more closely related to the dementia in A D than A/3P accumulation alone. However, it must be pointed out that dementia can occur in the absence of neocortical neurofibrillary tangles as demonstrated by Terry et

REFERENCES

al.62 In this regard, it is interesting to note that the H P N D cases may in fact be a preclinical form of A D as described by others 21'48'63. Price et al. 57 described a group of what they term "very mildly d e m e n t e d " cases which may resemble our H P N D cases. In that study, 25 cases were assessed p r e - m o r t e m using a 0 - 4 'Clinical Dementia Rating' (CDR). A C D R of 0 indicated no dementia, while a C D R value of '0.5' indicated 'very mild dementia', '1' indicated mild dementia, and so on. The C D R = 0.5 group (very mildly demented) exhibited extensive plaque and tangle formation similar to our H P N D and were described as being "cases which were at the threshold for clinical detection of dementia, even with very careful p r e m o r t e m assessment". In contrast, the C D R = 0 (nondemented) group exhibited tangles but no plaques and presented a pathologic profile similar to our NC cases. In contrast to these findings, however, Dickson et al. 24 report a group of subjects who were pathologically similar to our H P N D cases and exhibited no evidence of psychological impairment despite rigorous neuropsychometric testing. They concluded that these type of cases may not be 'preclinical A D ' . Thus, statements made concerning the pathologic correlates of dementia from patients such as the H P N D who have not been rigorously tested, psychometrically, can only be viewed as speculative. Finally, the present study confirms previous investigations of the distribution of plaques and tangles in the EC. These studies have all reported extensive tangle formation in layer II and what we describe as layer V, with fewer tangles in layers III and V112,31,64.

10

11

12

13 14

15

16

Acknowledgements. We wish to thank R. Brady, L. Sue, G. Binskin, J. Schaller and S. Mobley for technical assistance and Dr. H. Civin for neuropathologic evaluation. Supported in part by NIH Grants AG05344, AG08206 AG10688 and AG00295, the American Health Assistance Foundation, USPHS Grant NS 26146, Brain Bank Grant M H / N S 31862, and the Parkinson's Disease Foundation.

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