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					Tangles and Plaques in Nondemented Aging and “Preclinical” Alzheimer’s Disease
Joseph L. Price, DPhil,*† and John C. Morris, MD†‡§
The distribution and density of neurofibrillary tangles and amyloid plaques was studied in a unique series of cases whose premortem cognitive status had been assessed with the Clinical Dementia Rating (CDR), including 39 nondemented cases (CDR 0; age, 51– 88 years), 15 very mildly demented cases (CDR 0.5), and 8 severely demented (CDR 3) cases. The initial formation of tangles and plaques in healthy aging appeared to be independent of each other. Tangles were found in all the nondemented cases, especially in hippocampal and parahippocampal areas; the average tangle concentration increased exponentially with age. In contrast, plaques were absent in some brains up to age 88, and the earliest plaque formation in other cases occurred in the neocortex, in patches of diffuse plaques. Widely distributed neuritic as well as diffuse plaques throughout neocortex and limbic structures characterized a further group of nondemented cases. In these cases there was also a substantial increase over other nondemented cases, both in the number of tangles and in the rate of increase in tangles with age, suggesting an interaction between amyloid and neurofibrillary change at this stage. Such cases closely resemble CDR 0.5 cases, and it is proposed they represent “preclinical” Alzheimer’s disease. Price JL, Morris JC. Tangles and plaques in nondemented aging and “preclinical” Alzheimer’s disease. Ann Neurol 1999;45:358 –368

It is generally recognized that the incidence of Alzheimer’s disease (AD) is age related, increasing exponentially after the sixth decade of life.1 Neurofibrillary tangles and -amyloid plaques are commonly reported to occur in the brains of aged individuals, even without clinical criteria for AD.2– 4 Despite this, recent studies have suggested that when AD or other disorders are carefully excluded, aging itself may not result in substantial cognitive or neuronal loss.5–7 In contrast, even the mildest stage of dementing change may indicate the presence of AD, as supported by sufficient plaques and tangles for pathological diagnosis of the disease,8,9 and substantial neuronal loss in vulnerable areas such the entorhinal cortex6. To define the pattern of tangle and plaque formation during healthy aging, it is necessary to identify and study individuals without even very slight cognitive or memory impairment. We have an opportunity to study these issues, because of availability of a unique series of nondemented and very mildly demented cases who were assessed with the Clinical Dementia Rating (CDR).10 This instrument is sensitive enough to identify nondemented cases accurately, as well as to recognize the earliest clinically detectable stage of AD (CDR 0.5).8,11–13

In addition, it is very likely that there is a “preclinical” stage of AD, where the disease is present neuropathologically but has not produced any clinically detectable cognitive change. Because cases at the threshold for clinical detection of dementing change (CDR 0.5) already have large numbers of tangles and plaques, as shown by several previous studies,8,9,14,15 the initial development of AD and the earliest formation of tangles and plaques must occur before any detectable cognitive change. Well-characterized nondemented cases (CDR 0) may therefore be expected to divide into two groups, “healthy” aging cases without changes that are characteristic of AD, and preclinical cases with substantial ADrelated lesions. Recognition and characterization of a preclinical stage has important implications for treatment. If AD is well-established pathologically before it can be detected clinically, therapeutic interventions will need to focus on prophylaxis to be fully effective, as diagnosis may come too late to allow AD lesions to be reversed. Materials and Methods Selection and Collection of Material
The observations here were made on 39 nondemented cases without any indication of cognitive change (CDR 0; 21

From the Departments of *Anatomy and Neurobiology, ‡Neurology, and §Pathology, and †Alzheimer’s Disease Research Center, Washington University School of Medicine, St Louis, MO. Received Jul 21, 1998, and in revised form Nov 9. Accepted for publication Nov 9, 1998.

Address correspondence to Dr Price, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 S. Euclid Avenue, St Louis, MO 63110.

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male and 18 female), 15 cases with very mild dementia (CDR 0.5; 8 male and 7 female), and 8 severely demented cases (CDR 3; 4 male and 4 female). The age at death of the nondemented cases ranged from 51 to 93 years, including 26 cases age 75 and older and 13 cases younger than 75 (Table 1). The age at death of the very mildly demented cases ranged from 75 to 95, closely corresponding to the ages of the older nondemented group. The severely demented cases were age 62 to 95 years. None of the cases were members of AD kindreds with known genetic mutations. Those with dementia met clinical research inclusionary and exclusionary diagnostic criteria for dementia of the Alzheimer type that have a diagnostic accuracy rate for neuropathological confirmation of AD of 93%.13 The brains were also evaluated with a standard neuropathological battery; none of the demented cases had indications of Parkinson’s disease or other neurological or psychiatric disorders that might have contributed to the cognitive change. The apolipoprotein E genotype was available for 28 of the 62 cases studied. The occurrence of the ε4 allele was relatively high in the CDR 3 cases (8 of 12 alleles determined), but no other effect of the apolipoprotein E genotype was apparent. The CDR of each of the cases was determined by clinicians in the Memory and Aging Project of the Washington University Alzheimer’s Disease Research Center.10 A CDR score of 0 indicates no dementia, and CDR scores of 0.5, 1, 2, and 3 indicate very mild, mild, moderate, and severe dementia, respectively. The CDR score is derived from semistructured clinical interviews, and is assigned without reference to psychometric test results or to any postmortem neuropathological findings. Although CDR 0.5 has previously been characterized as “questionable” dementia, in the carefully characterized subjects enrolled for our studies the designation of CDR 0.5 is a dependable predictor of progression to more severe stages of dementia8,12 and correlates with histopathological AD.8,11,13 In these circumstances and with our highly selected sample, CDR 0.5 appears equivalent to very mild dementia. Two closely related assessment methods were used. The subjects of 40 cases were enrolled in a longitudinal study and were assessed before death using interviews with the subject and an informant. All subjects were assessed annually, and the last assessment was within a year of their death. At entry and every 2 years thereafter, the assessment was videotaped for independent review by a second clinician. In a few cases, there was disagreement about whether the CDR was 0 or 0.5 because of differences either between the two clinicians who assessed the interview or between the final and penultimate

0/0.5. assessments.11 Such cases were designated CDR The 40 cases assessed premortem included 22 CDR 0 cases, 5 CDR 0/0.5 cases, 6 CDR 0.5 cases, and 7 CDR 3 cases. Cognitive status was also assessed with a validated retrospective postmortem interview with an informant.16 For the 40 cases assessed premortem, this provided a CDR for the subject’s cognitive ability just before the terminal events leading to death. The same validated postmortem interview16 was used in an additional 23 cases (17 cases CDR 0; 2 cases CDR 0/0.5; 1 case CDR 0.5; and 3 cases CDR 3) from whom brain tissue was obtained from either the autopsy service of Barnes Hospital or the body donation program of the Department of Anatomy and Neurobiology of Washington University. In these cases, a CDR was assigned from the information provided by the interview. These cases were used to supplement the cases assessed premortem, especially for younger CDR 0 cases. Retrospectively assessed cases constituted 78% of younger CDR 0 cases (age 75 years), 25% of older CDR 0 cases (age 75 years), 20% of CDR 0.5 cases, and 43% of CDR 3 cases. Within each group, the age of the retrospectively assessed cases did not differ substantially from the age of the premortem assessed cases. In all cases designated CDR 0/0.5, plaques and tangles were present in sufficient densities for a neuropathological diagnosis of AD. The CDR 0/0.5 cases comprise 8 of the 15 very mildly demented cases in this series. Data from the CDR 0/0.5 and CDR 0.5 cases are presented separately in Figures 5 and 7. Because the histological findings on the two groups were so similar, however, the data were grouped together (as CDR 0.5) in other analyses.

Histological Procedures
The brains were fixed by immersion in buffered 10% formalin, cut into coronal slices about 1 cm thick, and then cut into smaller blocks for sectioning. In most cases blocks were taken from the ventral part of the cerebral hemisphere, from the posterior orbital cortex through the basal forebrain, the insula, and the entire temporal lobe. Sections were cut at 50 mm on a freezing microtome, and divided into series of 1 in 22 for staining. In all cases, series of sections were stained with a modified Bielschowsky reduced silver method optimized for fixed frozen sections.9 These sections were used for all of the quantitative analyses described below. Adjacent sections were stained by the Nissl method for correlation with brain cytoarchitecture. In addition, all observations were confirmed qualitatively with immunohistochemistry. Sections adjacent to the silverstained sections that had been selected for mapping and quantitative analysis (see below) were stained with antibodies against -amyloid (anti-A4 antiserum, gift of Dr Colin Masters; monoclonal antibody “Angela,” gift of Dr Dennis Selkoe; or monoclonal antibody “10D5,” gift of Athena Neurosciences), and with antibodies against paired helical filaments (monoclonal antibody “Pam,” gift of Dr Dennis Selkoe, or monoclonal antibody “PHF-1,” gift of Drs Sharon Greenberg and Peter Davies). In many cases, an additional series of sections was stained immunohistochemically with

Table 1. Subjects Grouped by CDR and Age CDR and Age Group CDR CDR CDR CDR CDR
CDR

Median Age (Range), yr 62 (51–73) 82 (75–93) 88 (75–95) 89 (75–95) 79 (62–95)

n 13 26 8 7 8

0, age 0, age 0/0.5 0.5 3

75 yr 75 yr

Clinical Dementia Rating.

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the Alz-50 antibody (gift of Dr Peter Davies). Although the Bielschowsky silver stain was the most consistent across all the cases studied, and was therefore used for the quantitative analyses, in many cases the immunohistochemical stains (especially with antibody 10D5 for amyloid and antibody PHF-1 for paired helical filaments) were more sensitive. In a few cases, apparent plaques could not be confirmed with the immunohistochemical stains; on careful examination, these were found to be artifacts of the silver stain.

nal cortical areas and field CA1), but plaques were found in neocortical areas. Because of these differences in both incidence and distribution, tangles and plaques are described in separate sections. In a third section, possible interactions between plaques and tangles are considered. Tangles
NONDEMENTED CASES (CDR 0)

Methods of Analysis
Amyloid plaques, as demonstrated by the Bielschowsky silver method, were divided into two types, diffuse and neuritic. Diffuse plaques were flocculent depositions of amyloid, without any inclusion of thickened, dark-staining neurites, or a dense central core. They ranged from small, rounded structures to large, irregular complexes. Any stained neurites running through these plaques were normal in diameter and appearance. Neuritic plaques were defined as those with thickened, darkly stained, and often contorted neurites within or extending from the region of amyloid deposition, and/or with a dense -amyloid core. Double immunohistochemical staining with two antibodies (one against amyloid and one against paired helical filaments) was used in some brains to confirm that the dystrophic neurites within neuritic plaques contain the same paired helical filaments found in tangles. For purposes of analysis, no distinction was made between intracellular and extracellular tangles. The distribution of tangles, plaques, and immunoreactive cells was mapped from silver-stained sections with the aid of a computerized microscope digitizer (Minnesota Datametrics, St Paul, MN), as described previously.9 An accessory computer program allowed the number and density of markers to be measured within defined areas. The structures sampled included the anterior olfactory nucleus and other olfactory cortical areas, the nucleus basalis of Meynert, the amygdaloid nuclei, the entorhinal cortex and other parts of the hippocampal formation, the posterior orbital cortex, and most of the insular and temporal cortex. Depending on differences in tissue availability and structure size, each structure was sampled from one to four times in each brain. To compare the patterns of tangle distribution between cases with different numbers of tangles due to age or level of dementia, areas were ranked in order of tangle density within each brain. The area with the highest tangle density was rated first, and so on. Areas with the same tangle density (eg, areas that had no tangles) were given the same, averaged rank.

Variation in tangle density with age. In the nondemented cases younger than 75 years, there were relatively small numbers of tangles, although at least a few tangles were found in all cases, especially in the perirhinal and entorhinal cortex (Fig 1) and in the anterior olfactory nucleus. Other limbic areas, including field CA1 of the hippocampus, consistently had many fewer, if any, tangles in these younger cases. There were almost no tangles in the neocortex or in the nucleus basalis of Meynert. In the nondemented cases aged 75 years or older, there were greater numbers of tangles but the distribution was very similar to that in the younger cases (see Fig 1). Many tangles were again found in the entorhinal and perirhinal cortex and, in contrast to the younger cases, there were also many tangles in hippocampal field CA1. A few tangles were found in the neocortex, especially in the inferior temporal cortex. The age-related increase in tangles in nondemented cases is exponential (Fig 2A), indicating that the rate of tangle formation increases with age, especially after age 70. There is also an increase in variability with age. Some of the older cases had large numbers of tangles, but others had low tangle densities that were comparable with those in younger cases. Although some of the nondemented cases also had amyloid plaques that might have influenced tangle formation, the same pattern of tangle density with age was also seen in 21 nondemented cases without plaques (although there is an increase in the average number of tangles in cases with plaques; see below) (see Fig 2B). Thus, a highly significant, exponential increase in tangle density occurs in nondemented aging, with or without plaques. Pattern of distribution of tangles. As described in Materials and Methods, a rank order analysis was used to compare the patterns of distribution of tangles in brains with different ages. As expected, this analysis showed that the distribution of tangles was very similar in younger and older groups of cases, despite the marked differences in the number of tangles at different ages (Fig 3). The entorhinal cortex, perirhinal cortical area 35, and the anterior olfactory nucleus had the highest concentrations of tangles and there was a much lower density of tangles in hippocampal field CA3, the subiculum, the piriform cortex, the nucleus basalis of

Results As observed previously from a smaller set of cases,9 the pattern of tangle formation in aging nondemented brains is very different from that of plaques. At least a few tangles were found in vulnerable areas of all aging brains, beginning in at least the sixth decade of life. Plaques, in contrast, were found in only a fraction of nondemented cases and were absent from some brains even in the late 80s. Furthermore, tangles occurred preferentially in specific parts of the hippocampus and related structures (especially the entorhinal and perirhi-

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Fig 1. Maps of neurofibrillary tangles from sections through the temporal lobe of 3 CDR 0 cases of different ages. Each dot represents one tangle. None of these cases had any plaques. The number of tangles increases with age, although the pattern of distribution is about the same. CA numbers hippocampal fields; other numbers Brodmann areas (20 inferior temporal cortex; 21 middle temporal cortex; 22 superior temporal cortex; 35 perirhinal cortex; 36 perirhinal cortex); Ins insula; SN substantia nigra; DG dentate gyrus; PSub presubiculum and parasubiculum; Sub subiculum; EC entorhinal cortex; Ca caudate nucleus; Re reticular nucleus of thalamus; VL ventrolateral nucleus of thalamus; P putamen; MD mediodorsal nucleus of thalamus; CeM centromedian nucleus of thalamus; GPe globus pallidus, external division; GPi globus pallidus, internal division. CDR Clinical Dementia Rating.

Fig 2. Graphs of the density of tangles in three limbic areas, as a function of age in nondemented (CDR 0) cases. The open symbols represent values for each area from individual brains. The lines connecting the filled symbols represent the average value for all cases within each 5 years of age. (A) Data from all cases rated CDR 0. Spearman rank correlation: r 0.78, 0.77, and 0.70, for the entorhinal cortex (EC), perirhinal cortex (A35), and CA1, respectively; p 0.001, for all three areas. (B) Data from only those cases in which no amyloid plaques were detected with silver or immunohistochemical stains. Spearman rank correlation: r 0.79, 0.70, 0.77 for the entorhinal cortex, perirhinal cortex, and CA1, respectively; p 0.001, for all three areas. Note that although the younger cases all had tangles, they were too few to register on this scale. CDR Clinical Dementia Rating.

Meynert, and the neocortical areas. The only significant difference between younger and older cases was that hippocampal field CA1 and the periamygdaloid cortex developed tangles at relatively older ages. Thus, field CA1 and the periamygdaloid cortex had few tangles in the younger cases but ranked among the most affected areas in the brains of those cases aged 75 years or older (see Fig 3). The apparent difference in neocortical areas (temporal cortical areas 20, 21, and 22, and the orbital and insular cortex) was due to the virtual absence of tangles in the neocortex in younger brains, such that no distinctions were apparent between them. As tangles developed in these areas in the older

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VERY MILDLY DEMENTED CASES (CDR

0/0.5 OR 0.5).

No difference was found in the number or distribution of tangles in CDR 0/0.5 and CDR 0.5 cases. Both groups of very mildly demented cases had more tangles, distributed over a wider region than found in most of the older nondemented cases (Fig 4), although there was overlap in individual cases (see below; see Fig 7). The very mildly demented cases consistently had high tangle densities in vulnerable areas such as the entorhinal or perirhinal cortex, with lower numbers of tangles in the neocortex. The increase in tangle density with age seen in nondemented cases was not found in the very mildly demented cases, presumably because the age effect has been overwhelmed by a disease effect in these cases. As shown by the rank order comparison, the qualitative pattern of distribution of tangles in CDR 0.5 cases was the same as in the nondemented cases (see Fig 3B).
SEVERELY DEMENTED CASES (CDR 3). In the severely demented cases, the density of tangles increased further over that seen in very mildly demented cases, although there was still some overlap between individual cases (see Fig 8). The patterns of distribution of tangles in the CDR 3 cases, as shown by the rank order analysis, were very similar to that in the CDR 0 and CDR 0.5 cases (see Fig 3B). The only noteworthy difference was that the much greater number of tangles throughout many parts of the brain decreased the variation between areas.

Fig 3. Graphs illustrating the distribution of tangles across 17 brain areas, expressed as the rank order of each area (see Materials and Methods). Those areas with the highest tangle density within each brain are ranked near 1, areas with progressively fewer tangles had numerically larger ranks. The rank orders are averaged across two age groups (age 75 years and age 75 years) in A, or across CDR groups (CDR 0, CDR 0.5, and CDR 3) in B. The error bars represent SEM values. The patterns are similar, despite that the number of tangles increases markedly across the age and CDR groups (see text). CA numbers hippocampal fields; A numbers Brodmann areas (A20 inferior temporal cortex; A21 middle temporal cortex; A22 superior temporal cortex; A35 perirhinal cortex; A36 perirhinal cortex); Sub subiculum; EC entorhinal cortex; Orb orbital cortex; Ins insular cortex; TP temporal polar cortex; AON anterior olfactory nucleus; PC piriform cortex; PAC periamygdaloid cortex; Amyg amygdala; SI substantia innominata (nucleus basalis of Meynert).

Plaques
NONDEMENTED CASES (CDR 0). Substantial differences in the number and distribution of plaques were found in the nondemented cases. Because these differences were very distinct, the nondemented cases were most readily analyzed in three groups (A, B, and C), based on the qualitative differences in the type, and distribution of plaques (Table 2). Although the three groups also differ in the number of plaques, quantitation of plaques is uncertain because of their large variation in size and shape. The use of clear, qualitative criteria allows stages in the development of plaques to be distinguished that are independent of precise numbers. Group A had no plaques in any part of the forebrain, in sections stained either with the modified Bielschowsky silver method or with antibodies against -amyloid. This group consisted of the 13 younger cases (age, 51–73 years) and 8 of the 26 older nondemented cases (age, 75– 88 years) (see Table 2). Group B consisted of 11 of the 26 cases aged 75 years or older (see Table 2). These cases had relatively few amyloid plaques, all of which were diffuse in type and were distributed in restricted patches in the neocortex (Figs 5 and 6A–C). Across the different brains, the patches

brains, it became apparent that the inferior temporal area 20 and the orbital and insular cortex had more tangles than the middle and superior temporal areas 21 and 22. Even with these differences, the correlation between the rank order values for the two age groups was highly significant (linear correlation coefficient r 0.85, p 0.001).

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Fig 4. Maps of neurofibrillary tangles and amyloid plaques in a section through the temporal lobe from a typical CDR 0.5 case. Each small triangle (on the left side) represents one tangle, and each dot (on the right) represents one plaque. Diffuse plaques are shown by small dots, while neuritic plaques are designated by larger dots. Tangles are concentrated in the entorhinal cortex, perirhinal cortex (area 35), and hippocampal field CA1, and plaques are most concentrated in neocortical areas 36, 20, and 21. Numbers Brodmann areas (20 inferior temporal cortex; 21 middle temporal cortex; 22 superior temporal cortex; 35 perirhinal cortex; 36 perirhinal cortex); CA1 hippocampal field CA1; Sub subiculum; PSub presubiculum and parasubiculum; EC entorhinal cortex.

Table 2. Nondemented (CDR

0) Subjects Grouped by Plaque Type Median Age (Range), yr n 13 8 11 7 Plaque Type Number None None Few Many Distribution

Group Group Group Group
CDR

A, age A, age B C

75 yr 75 yr

63 (51–73) 84 (75–88) 83 (75–92) 78 (75–93)

Diffuse only Neuritic/diffuse

In patches Extensive

Clinical Dementia Rating.

varied from a few small areas with relatively few plaques to more extensive areas with a higher plaque density. The patches tended to be located within sulci, but they were not consistently concentrated in any given cortical region. In the third group (Group C), made up of 7 of the 26 cases older than 75 (see Table 2), there were neuritic plaques as well as diffuse plaques, distributed extensively in large numbers across the neocortex (see Figs 5 and 6D). In addition, there were also many plaques in limbic structures, especially the entorhinal and perirhinal cortex. In the neocortex, most plaques were diffuse in type, but in limbic structures, a much higher percentage of plaques were neuritic (see Fig 5). In these respects, cases in Group C resembled the very mildly demented cases (CDR 0.5, see below). Although the mean density of neocortical plaques in Group C was lower than in CDR 0.5 cases, there was overlap between individual cases. As discussed below, the cases in Group C are presumed to be preclinical cases of AD.

VERY MILDLY DEMENTED CASES (CDR

0/0.5 AND 0.5).

The very mildly demented cases had substantial numbers of both diffuse and neuritic plaques distributed widely throughout the cerebral cortex (see Fig 4). There was little or no difference between the CDR 0/0.5 and CDR 0.5 groups (see Fig 5). Most plaques in the neocortex were diffuse in type, although there was a sizable number of neuritic plaques. The limbic areas, especially the hippocampus, had relatively fewer plaques than the neocortical areas (see Figs 4 and 5). The plaque density in the amygdaloid nuclei and the entorhinal and perirhinal cortex was moderate, but the average plaque density in hippocampal field CA1 and the subiculum was less than onethird of that in the temporal, insular, or orbital cortex. As in the nondemented Group C, the proportion of plaques that were neuritic was substantially higher in the limbic areas than in the neocortex (see Fig 5).
SEVERELY DEMENTED CASES (CDR 3). In severely demented cases, there was little if any change in the total

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Fig 5. Graphs of plaque density in the orbital (Orb), insular (Ins), and temporal neocortex (Temp) (A) and in the amygdala (Amyg), perirhinal cortex (area 35, A35), entorhinal cortex (EC), and hippocampal field CA1 (B). The CDR 0 cases are divided into the following three groups: Group A, with no plaques (subdivided between cases age 75 years and cases age 75 years); Group B, with only diffuse plaques; and Group C, with neuritic and diffuse plaques. The line depicts the percentage of plaques that are neuritic in each group.

density of plaques over those seen in very mildly demented cases, with almost complete overlap between individual cases (see Fig 5). There was a shift in the type of plaques, however, to an increased proportion of neuritic plaques and a commensurate decrease in the proportion of diffuse plaques, especially in the neocortex. Tangle Formation as a Function of Plaque Density The description above indicates that the earliest formation of tangles is separate from that of plaques. Observations in Down syndrome and familial forms of AD in which changes in -amyloid and amyloid precursor protein result in neurofibrillary as well as amyloid changes,17 however, strongly suggest that there is interaction between amyloid deposition and neurofibrillary changes at a subsequent stage. The density of tangles in the entorhinal cortex, perirhinal cortical area 35, hippocampal field CA1, and the temporal neocortex of

nondemented cases was therefore analyzed as a function of Groups A, B, and C (Fig 7). This analysis showed that there is an increase in the age-related accumulation of tangles in cases with substantial numbers of plaques. As expected from the description above, the younger cases in Group A (age, 75 years) had relatively few tangles in the entorhinal cortex, perirhinal cortical area 35, and the hippocampal field CA1, and virtually none in the temporal cortex (see Fig 7). In keeping with the increase in tangles with age (described above), there were significantly more tangles in the older cases ( 75 years), either those with no plaques (Group A) or those with patches of diffuse plaques (Group B). The presence of diffuse plaques in Group B was associated with a slight but not significant increase in average tangle density over the older cases in Group A. On the other hand, in Group C, the substantial number of neuritic and diffuse plaques was associated with a substantial and significant increase in tangle density in both limbic areas and the temporal neocortex. Approximately the same density of tangles was found in the very mildly demented cases (CDR 0/0.5 and CDR 0.5), with no significant difference between Group C and the CDR 0/0.5 or CDR 0.5 groups (see Fig 7). In the severely demented cases (CDR 3), there was a further, significant increase in tangle density. By plotting tangle density versus age for Groups A, B, and C, it was also possible to analyze the interaction between plaque deposition and the age-related rate of formation of tangles (Fig 8). The slope of a linear regression line fitted to these plots provided an estimate of the increase in tangles per year of age (see Fig 8A). As expected from the results described above, among the cases with no plaques (Group A), there was a higher rate of increase in tangle density with age in the older cases ( 75 years) than in the younger cases, both in limbic structures (the entorhinal cortex, perirhinal cortical area 35, and the hippocampal field CA1) and in the temporal neocortex (see Fig 8B). The rate of increase in tangle density was slightly greater in the cases with patches of diffuse plaques (Group B). In Group C, with many more plaques, there was a marked increase in the rate of tangle accumulation. This suggests that the deposition of amyloid plaques accelerates the age-related formation of tangles. Discussion These observations on carefully assessed nondemented cases indicate that the initial formation of tangles is separate from that of plaques, both spatially and temporally. Tangles form preferentially in the limbic structures, and plaques form first in the neocortex. At least a few tangles were found in all brains examined, and the average tangle density increases with age, even in the absence of plaques. In contrast, plaques were not

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Fig 6. Maps of the distribution of amyloid plaques in sections through the temporal lobe of 4 CDR 0 cases. (A, B, and C) Representative cases from Group B, with patches of diffuse plaques in parts of the temporal cortex. The case in A has only a very small patch of plaques, whereas B and C have larger, multiple patches. (D) Representative case from Group C, with widespread neuritic and diffuse plaques. Small dots represent diffuse plaques, and the larger circles represent neuritic plaques. CA numbers hippocampal fields; other numbers Brodmann areas (35 perirhinal cortex; 36 perirhinal cortex); Sub subiculum; PSub presubiculum and parasubiculum; EC entorhinal cortex.

found in some cases as old as age 88. We conclude that the initial formation of tangles and the earliest amyloid deposition in plaques are independent events. Subsequently, however, there appears to be interaction between amyloid and neurofibrillary changes. The deposition of large numbers of plaques in a subset of nondemented cases (Group C) is associated not only with the development of dystrophic neurites within neuritic plaques, but also with an increase in both the number of tangles and the rate of tangle formation with age. Because the pattern of distribution of tangles is the same with or without plaques, the apparent effect of amyloid is to accelerate an age-related tauopathy, which is otherwise relatively slow. We propose that the nondemented cases with neuritic plaques (Group C) represent preclinical AD. The existence of a preclinical group must be postulated because the CDR 0.5 cases, at the threshold of very

mild dementia, all contain large numbers of tangles and plaques.8,9 Because these lesions accumulate relatively slowly, the disease process must begin at an earlier stage, before clinical detection is possible. As expected for preclinical AD cases, the cases in Group C closely resemble CDR 0.5 cases in the number and type of plaques and tangles. As recognized by the CERAD (Consortium to Establish a Registry for Alzheimer’s Disease) criteria for diagnosis of AD,18,19 neuritic plaques provide an especially good indicator of AD, because they represent the conjunction of amyloid deposition and neurofibrillary change. Relation to Other Studies An important aspect of this study was that the premortem cognitive status of each case was carefully assessed clinically. Recognition of very mildly demented cases (CDR 0.5) assured that the CDR 0 cases were

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Fig 7. Graphs of tangle density in hippocampal field CA1, the entorhinal cortex (EC), the perirhinal cortex (area 35, A35), and the temporal neocortex (Ctx), in cases grouped by CDR or within CDR 0, into plaque Groups A, B, and C. There is a highly significant increase in tangle density for all brain areas between the younger and older cases in Group A, between Group B and Group C, and between CDR 0.5 and CDR 3 cases, but there is no significant difference between 0.1, for all comparisons the other groups (**p 0.001; #p between the same areas in adjacent groups; one-tailed t test). Note the large increase in the tangle density in the temporal cortex in the CDR 3 cases. Fig 8. (A) Graph of tangle density against age in the entorhinal cortex in CDR 0 cases. The cases are divided into Groups A, B, and C according to the presence or type of plaques found, and Group A is subdivided into younger and older cases. For each set of cases, a “best fit” linear regression line was calculated and drawn. The slope of this line, representing the increase in tangle density with age, increases between young and old cases without plaques (Group A), and between Group B and Group C. (B) Graphs of the rate of increase in tangle density with age calculated from the slope of linear regressions for Groups A, B, and C. There is an increase in the rate of tangle formation both with age (between younger and older cases in Group A) and with increasing plaque deposition (between cases with age over 75 years in Groups A, B, and C). EC entorhinal cortex; CA1 hippocampal field CA1; A35 Brodmann area 35, perirhinal cortex; Temp temporal neocortex.

truly free of cognitive change. In a smaller previous study that used the CDR,15 the authors also speculated that 2 of 15 CDR 0 cases containing substantial numbers of neuritic and diffuse plaques represented preclinical AD. In a recent large study (2,661 cases), Braak and Braak20 analyzed stages of AD-related lesions in relation to age, based on a previous study21 that defined three neuropathological stages of amyloid deposition and six stages of neurofibrillary changes. In this study, the cognitive status of the cases was not carefully determined, and the amyloid and neurofibrillary stages are not closely related to each other or to the presence or level of dementia. Despite this, the spatial patterns and progression of amyloid and neurofibrillary changes that underlie these stages are very similar to those described here and previously.9 An important observation in the study by Braak and Braak20 was that neurofibrillary changes and plaque deposition may begin at young ages, even before age 30. Subsequently, the prevalence of both markers steadily increases with age. There are several differences between the detailed results of the study by Braak and Braak20 and the present observations, but there is also considerable agreement in important respects. In separate commentaries, Duyckaerts and Hauw22 and Silverman and colleagues23 calculated from the data of Braak and Braak20 that the

incidence of neurofibrillary stages I/II would begin virtually at birth and would reach 50% prevalence at about age 48. The earliest amyloid stage reached 50% prevalence 25 years later, at about age 73. The important conclusion from both these results and the present study is that the earliest neurofibrillary changes occur well before the earliest amyloid deposition. The calculations also indicate that there is a 35- to 40-year gap between earliest neurofibrillary stage I/II and the intermediate stage III/IV,22,23 indicating that early neurofi-

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brillary changes develop very slowly. Further, the development of amyloid occurs before the later, more severe neurofibrillary stages (Braak amyloid stages A and B rise in prevalence before neurofibrillary stages III/IV, and the late Braak amyloid stage C occurs before the late neurofibrillary stages V/VI). In agreement with the present results, therefore, these observations indicate that mild neurofibrillary changes begin before amyloid deposition, but severe neurofibrillary changes occur only after amyloid deposition. This supports the hypothesis presented here that amyloid deposition exacerbates the mild neurofibrillary changes seen in aging. What Marks the Beginning of AD? The early and virtually ubiquitous formation of tangles has led to the suggesting that tangles represent the earliest stage of AD (eg, by Braak and Braak20). If this is the case, AD is inevitable and may begin in the first decades of life. Tangle formation is closely correlated with cell loss, and there is considerable opinion that any tangle formation reflects pathological change.24 For example, the early formation of tangles in the entorhinal cortex closely parallels cell loss in very mildly demented (CDR 0.5) cases.6 In contrast, in the superior temporal sulcus where plaques develop early in AD but tangles are formed later, cell loss occurs only in late AD.25,26 Furthermore, the recent genetic observations in frontotemporal dementia and parkinsonism linked to chromosome 17 indicate that genetic alterations in tau by itself can result in neurodegeneration and dementia.27,28 Tangle formation during nondemented aging does not appear to be associated with age-related cell loss, however. Despite tangles in the entorhinal cortex and hippocampal field CA1, no cell loss has been identified in these structures as a function of nondemented aging.5,6 Although this may be related to the difficulty of recognizing small amounts of cell loss, it would appear that age-related tangle formation is too slow to produce significant cell death during current life spans. We conclude that age-related tangle formation, although a mild form of tauopathy, does not by itself represent a process that would inevitably progress to AD. In contrast, several observations in Down syndrome and familial forms of AD indicate that genetic factors resulting in changes in -amyloid are sufficient to cause AD, including tangles and other neurofibrillary changes.17 These observations provide strong evidence that -amyloid can produce or exacerbate neurofibrillary changes, at least after the initial stages of amyloid deposition. Furthermore, the most distinct indication of the onset of dementia (in CDR 0.5 cases) is a substantial increase in plaques.11 As discussed above, most CDR 0 cases do not have large numbers of plaques, and the small number of nondemented cases

with substantial numbers of plaques can be recognized as preclinical AD. If, as indicated by the present data, extensive development of plaques accelerates age-related tangle formation, then amyloid deposition presumably converts the ubiquitous but slow neurofibrillary changes during aging into a disorder that leads inexorably to the severe stages of AD. The mechanisms by which -amyloid might affect tangle formation are unclear but may include disturbances in calcium homeostasis, inflammatory reactions, and oxidative stress. Finally, it should be asked whether the patches of diffuse amyloid plaques that are seen in pathologically less affected nondemented cases represent a still earlier stage in AD. Such plaques are not seen in all aging cases, at least well into the ninth decade, but where they do occur, a progression can be recognized from cases with few, very small patches of diffuse plaques to cases with more numerous and larger patches. The preclinical cases with extensive plaques, both neuritic and diffuse, appear to represent the continuation of this progression. Note Added in Proof
Another study published since this paper was accepted for publication has also identified preclinical AD cases neuropathologically from 31 nondemented aged subjects (CDR 0; age range, 72–102 years), based on the presence of neuritic plaques (CERAD “Possible AD”).29 Tangles were not used as the defining characteristic, and all the cases studied had some tangles (at least Braak stage 1). As a group, the preclinical cases had substantially more tangles and other neurofibrillary changes (average Braak stage, 2.3) than “normal” aging cases (average Braak stage, 1.3). These results agree well with the observations presented here.
Supported by NIH grants AG03991 and AG05681. We thank, in particular, Dr Leonard Berg for his inspiration, help, and guidance. We thank Hieu Van Luu for excellent histological preparation of the sections, and Melissa Rundle and Karan Randhava for dedicated computer mapping and analysis of tangles and plaques. We also thank Drs Eugene Rubin, Martha Storandt, and the other physicians and staff of the Memory and Aging Project and the Clinical Core of the Alzheimer’s Disease Research Center (ADRC) of Washington University for subject evaluations, Dr Dan McKeel and the staff of the Neuropathology Core of the ADRC for help in obtaining brain tissue, and Phillip Miller and Elizabeth Grant of the Biostatistics Core of the ADRC for statistical and database assistance.

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