Original article
Spatial patterns of β-amyloid (Aβ) deposits in familial and sporadic Alzheimer’s disease

Introduction

The neuropathology of Alzheimer’s disease (AD) is characterised by the formation of extracellular se­nile plaques (SP) and intracellular neurofibrillary tangles (NFT) [8]. The most important molecular consti­tuent of the SP is -amyloid (A) [27], a peptide of 36-43 amino acids arising by constitutive cleavage of a trans-membrane glycoprotein amyloid precursor pro­tein (APP). A variety of A peptides are formed as a result of secretase cleavage of APP [31]. The most common of these peptides is A42, cleaved in the trans-Golgi network and found largely in discrete A deposits, whereas the more soluble A40, cleaved in the endoplasmic reticulum, is also found in association with blood vessels [41,45] and may develop later in the disease [23]. Oligomers that form on the amyloid pathways may be cytotoxic species rather than A42 [37]. The discovery of -amyloid (A) led to the formulation of the ‘amyloid cascade hypothesis’ (ACH), the most important model of the molecular pathology of AD developed over the last 20 years [32]. Essentially, the ACH proposes that the deposition of A is the initial pathological event in AD leading to the formation of NFT, cell death, and ultima­tely dementia.

At least four genetic loci have been implicated in AD, viz., the APP gene on chromosome 21 [20,28], the presenilin (PSEN) genes on chromosomes 14 (PSEN1) [48] and 1 (PSEN2) [38], and the apolipoprotein E (APOE) gene on chromosome 19 [47]. Mutations in APP and PSEN genes may alter APP meta­bolism, resulting in increased deposition of A, while allelic polymorphism of APOE, and especially the expression of the 4 allele, may increase the proportion of the more fibrillogenic A42 formed in the tissue [18,19,25]. These genetic factors, however, may not explain the majority of AD cases [30]. Hence, early-onset AD linked to APP and PSEN mutations may account for less than 5% of the total number of cases [34]. Additional susceptibility genes and environmental factors are therefore likely to be involved in AD, especially in sporadic cases. In isolated Amish communities, for example, 24 markers have been linked to dementia [40], and several other linkage studies have shown the presence of possible AD-related genes on chromosomes 9, 10, and 12 [49].

Three morphological subtypes of A deposit are observed in histological preparations of AD brain [5,22]: (1) diffuse (‘pre-amyloid’) deposits, in which the A peptide is not aggregated into amyloid and dystrophic neuritis (DN) and paired helical filaments (PHF) are infrequent or absent, (2) primitive (‘neuri­tic’) deposits, in which the A is aggregated into amyloid and is associated with DN and PHF, and (3) classic (‘cored’) deposits, in which A is highly aggregated to form a central amyloid ‘core’ surrounded by a ‘ring’ of DN. Diffuse A deposits are often spatially correlated with neuronal perikarya [1,6] and may represent the earliest stages of A deposition in AD [5]. Different A deposit subtypes could represent stages in the maturation of a single deposit type [5]. Hence, diffuse deposits may evolve into more mature deposits as the disease progres­ses [5].

A deposits are often clustered in the cerebral cortex, the clusters being distributed in a regular pattern parallel to the pia mater [13,15,17], and may be the result of A pathology developing in relation to the cortico-cortical and cortico-hippocampal pathways [24,44]. Hence, either toxic A oligomers cause degeneration of specific cortico-cortical pathways or degeneration of cortico-cortical pathways results in the deposition of A [13]. The objective of this study was to determine whether the spatial pattern of A deposits in the cortex was influenced by genetic factors. Hence, the spatial patterns of diffuse, primitive, and classic A deposits were studi­ed in three groups of AD cases: (1) early-onset familial AD (EO-FAD) linked to mutations of APP or PSEN1, (2) late-onset FAD (LO-FAD) not linked to APP or PSEN genes, and (3) cases of SAD. In addition, the influence of APOE genotype on the spatial patterns of A deposits was studied.

Material and methods

Cases

Eighteen cases of AD (details in Table I), obtained from the Brain Bank, Department of Neuropathology, Institute of Psychiatry, King’s College, London, UK, were studied. Informed consent was given for the removal of all brain tissue according to the 1996 Declaration of Helsinki (as modified Edinburgh, 2000). Patients were clinically assessed and all fulfilled the ‘National Institute of Neurological and Com­municative Disorders and Stroke and the Alzheimer Disease and Related Disorders Association’ (NINCDS/ADRDA) criteria for probable AD [52]. The histological diagnosis of AD was established by the presence of widespread neocortical SP consistent with the ‘Consortium to Establish a Registry of Alzheimer Disease’ (CERAD) criteria [42] and ‘Natio­nal Institute on Aging (NIA)-Reagan Institute’ criteria [36,43]. The cases were divided into three groups: (1) EO-FAD (onset ≤ 65 yrs) (N = 4); two cases linked to the APP717 mutation and two to PSEN1 mutations (G209V and E280A), (2) LO-FAD (≥65 yrs) (N = 5) not associated with mutations of APP or PSEN genes, and (3) SAD (N = 9) with no evidence of familial involvement. The EO-FAD cases expressed APOE genotype 2/3 or 3/3, while two of the LO-FAD cases expressed APOE genotype 3/4. Of the SAD cases, four expressed APOE genotype 3/4, one case 4/4, and the remaining cases expressed genotype 2/3 or 3/3.

Tissue preparation

Blocks of the frontal and temporal cortex were taken at the level of the genu of the corpus callosum and lateral geniculate body respectively to study the superior frontal gyrus (SFG), superior temporal gyrus (STG) and the parahippocampal gyrus (PHG). Tissue was fixed in 10% phosphate-buffered formal saline and embedded in paraffin wax. 7 m coronal sections were stained with a rabbit polyclonal antibody (Gift of Prof. B.H. Anderton, Institute of Psychiatry, King’s College London) raised to the 12-28 amino acid sequence of the A protein [50], and which distinguishes the major types of A deposit [5]. The antibody was used at a dilution of 1 in 1200 and the sections incubated at 4°C overnight. Sections were pre-treated with 98% formic acid for 6 minutes, which enhances A immunoreactivity. A was visua­lised using the streptavidin-biotin horseradish per­o­xidase procedure with diaminobenzidine as the chromogen. Sections were also stained with hae­matoxylin. A deposits were identified according to the criteria of Delaere et al. [22]: (1) diffuse deposits were 10-200 m in diameter, lightly stained, irregular in shape, and with diffuse boundaries; (2) primitive deposits were 20-60 m, well demarcated, symmetrical in shape, and strongly stained; and (3) clas­sic deposits were 20-100 m and had a distinct central amyloid core surrounded by a ‘corona’ of DN.

Morphometric methods

The spatial patterns of the A deposits were studied parallel to the pia mater in the upper 1 mm of the cortex using a magnification of × 100 [13]. A strip of cortex 17 600 to 25 600 µm in length, and which included a sulcus and a gyrus, was studied using 1000 × 200 m contiguous sample fields, the short dimension of the field being aligned with the surface of the pia mater [7]. Hence, the sample field included laminae I, II, and III. Between 64 and 128 contiguous sample fields were used to sample each gyrus. A micrometer grid with grid lines at intervals of 10 m was used as the sample field. The number of diffuse, primitive, and classic A deposits was counted in each field.

Data analysis

The data were analysed by spatial pattern analysis [2,10,13,14]. Essentially, the variance/mean (V/M) ratio of the data measures the degree of non-randomness present and determines whether A de­posits were distributed randomly (V/M = 1), regularly (V/M < 1), or in clusters (V/M > 1) along the strip of cortex parallel to the pia mater. The V/M ratio was calculated at increasing field sizes by adding together successively data from adjacent sample fields, e.g., 200 × 1000 m (the original field size) and then at 400 × 1000 m, 800 × 1000 m, etc., up to a size limited by the length of cortex sampled. The V/M ratio was plotted against the increasing field size to reveal the spatial pattern. If the deposits were clustered, then the analysis indicated whether the clusters themselves were randomly or regularly distri­buted and provides an estimate of the mean dimension of the clusters in a plane parallel to the pia mater.

The frequencies of the different types of spatial pattern (random, regular, regularly distributed clusters, large-scale clustering) were compared between groups using chi-square (2) contingency table tests. In addition, size of the clusters, measured parallel to the pia mater and averaged over all gyri, was compared between groups using two-factor analysis of variance (ANOVA) (STATISTICA software, StatSoft Inc., 2300 East 14th St, Tulsa, OK 74104, USA).

Results

The spatial patterns of the diffuse, primitive, and classic A deposits in the PHG of a single case (Case C, EO-FAD, PSEN1 mutation) are shown in Fig. 1. The V/M ratio of the diffuse deposits increased with field size without reaching a significant peak consistent with the presence of a large cluster of diffuse A deposits. The V/M ratio of the primitive deposits exhibited a significant peak at a field size of 200 m, suggesting the presence of clusters of pri­mitive deposits, 200 m in diameter, regularly distributed parallel to the pia mater. The V/M ratio of the classic deposits was significantly less than unity at all field sizes, suggesting a regular or uniform distribution of deposits.

The frequency of the different types of spatial patterns exhibited by the diffuse, primitive, and classic deposits in EO-FAD, LO-FAD, and SAD is shown in Table II. The diffuse deposits were distri­buted either in clusters which were regularly distri­buted parallel to the pia mater or in large clusters, no significant differences in spatial pattern being observed between groups. The primitive A de­posits, however, were more frequently distributed in regularly distributed clusters parallel to the pia mater in EO-FAD and LO-FAD and were more frequently distributed in large clusters in SAD (2 = 3.94, P < 0.05). In addition, the classic A deposits were less frequently distributed in regular clusters and more frequently distributed in large clusters in EO-FAD compared with LO-FAD (2 = 6.45, P < 0.05). The mean cluster sizes of the diffuse, primitive, and classic deposits, averaged over cortical regions, are shown in Fig. 2. The mean size of the diffuse deposits differed between groups (F = 3.64, P < 0.05), cluster size being significantly less in SAD compared with EO-FAD and LO-FAD. There were no significant diffe­rences in the cluster sizes of the primitive (F = 1.89, P > 0.05) or classic deposits (F = 0.66, P > 0.05) between groups. Within the EO-FAD group, there were no differences in the spatial pattern of the A deposits between APP and PSEN1 cases.

A comparison of the spatial patterns exhibited by the A deposits in cases classified according to APOE genotype is shown in Table III. There were no significant differences in the spatial patterns of the diffuse (2 = 0.02, P < 0.05), primitive (2 = 0.09, P < 0.05), or classic (2 = 6.16, P < 0.05) A deposits in cases expressing APOE genotypes 2/3 and 3/3, compared with those expressing genotypes 3/4 and 4/4. The mean cluster sizes of the diffuse, primitive, and classic deposits, averaged over the three cortical regions, are shown in Fig. 3. There were no significant differences in the cluster sizes of the diffuse (F = 0.27, P > 0.05) primitive (F = 2.45, P > 0.05) or classic deposits (F = 0.61, P > 0.05) between cases expressing different APOE genoty­pes, although there is some evidence that the cluster size of the classic deposits may be greater in cases expressing allele 4.

Discussion

The A deposits exhibited two common spatial patterns: (1) deposits were clustered with cluster sizes in the size range 200-3200 m in diameter, the clusters being regularly distributed parallel to the pia mater; and (2) deposits were clustered on a larger scale, usually greater than 3200 m in diameter, without regular spacing. These results are similar to those previously reported for A deposits [13,15,17] and for NFT [3,11] in AD.

Regular clustering of the diffuse and primitive deposits could be a consequence of degeneration of the cortico-cortical connections [15,24]. In the cerebral cortex, the cells of origin of the cortico-cortical projections are clustered and occur in bands which are regularly distributed along the cortex parallel to the pia mater. Individual bands of cells vary in width approximately from 400 to 1000 m depending on cortical area [33]. In some gyri, the estimated widths of the clusters of diffuse and primitive A deposits suggest an association with these projections [44]. By contrast, classic deposits often cluster around cerebral blood vessels, especially the vertically penetrating arterioles, which also exhibit a regular pattern of distribution parallel to the pia mater in laminae II/III [9]. By contrast, the large-scale clustering of deposits may be attributable to the aggregation of amyloid within cortical sulci [13]. The density of both neurons and blood vessels is greater in sulci compared with the gyral crest, which could explain these accumulations [4,26].

There were no significant differences in the frequency of the different types of spatial pattern of the diffuse deposits either between EO-FAD, LO-FAD, and SAD, or between cases classified according to APOE genotype. Diffuse deposit clusters, however, were larger in EO-FAD and LO-FAD than in SAD, suggesting that initial deposition of A is greater in FAD and that a higher proportion of diffuse deposits may be converted to primitive deposits in SAD [5].

Clusters of primitive deposits were more frequently regularly distributed in EO-FAD and LO-FAD and more frequently present in non-regularly distributed clusters in SAD. Hence, in FAD, conversion of diffuse to primitive deposits may result in a more localized and specific spatial pattern of deposits in relation to the cortico-cortical pathways. By contrast, in SAD, a greater proportion of diffuse deposits may be converted to primitive deposits, resulting in clusters of primitive deposits of similar size to those of FAD and clusters of diffuse deposits smaller than those of FAD.

Classic deposits were less frequently distributed in regular clusters and more frequently present in larger clusters in EO-FAD. Previous studies suggest that classic deposits are more likely to be clustered around blood vessels in SAD than in FAD [12]. Classic deposits also occur with greater density in AD cases with extensive capillary amyloid angiopathy (CAA) [16]. CAA is more frequent in cases with specific gene mutations, APP692 mutations, for example, re­sulting in extensive CAA and numerous large-cored deposits clearly distinct in morphology from those of SAD [21,46]. Hence, in cases with APP and PSEN mutations, the blood-brain barrier may be significantly damaged, increasing diffusion from blood ves­sels, and influencing the formation of classic de­posits at greater distances from blood vessels than in LO-FAD and SAD.

All types of A deposit exhibited similar spatial patterns and cluster sizes in cases classified according to APOE genotype. APOE genotype has been identified as a major risk factor in AD, individuals with the disease having 2-3 times the frequency of allele 4 compared with non-demented elderly controls [51]. The presence of allele 4 may accelerate the development of AD pathology within the aged brain, and hence is often associated with an earlier disease onset [29]. In addition, the majority of studies report increased amyloid deposition in individuals expressing allele 4 [18,19,25]. The present data suggest, however, that APOE genotype had little effect on the spatial pattern of the A deposits in the frontal and temporal lobe. Some studies suggest that A deposition, specifically in the form of A40, may be more closely related to APOE genotype [35,39]. Hence, it would be of interest to study the spatial patterns of A40 and A42 deposits separately in relation to APOE genotype.

In conclusion, the data suggest that gene expression had relatively little effect on the type of spatial pattern exhibited by the diffuse A deposits. The aver­age cluster size of the diffuse deposit, however, was larger in FAD compared with SAD, suggesting greater initial A deposition in FAD. However, cluster sizes of the primitive deposits were similar in FAD and SAD, suggesting that (1) a higher proportion of diffuse deposits may be converted to primitive deposits in SAD, and (2) primitive deposits may be more closely related to specific cortico-cortical pathways in FAD. Classic deposits were more extensively distributed in the cortex in FAD than SAD. The presence of the APOE allele 4 had little effect on the spatial patterns or cluster sizes of A deposits.

Acknowledgments

Brain tissue sections were kindly provided by the Brain Bank, Dept. of Neuropathology, Institute of Psy­chiatry, King’s College London, UK. We would like to thank Mrs Mavis Kibble and Mr Alan Brady for technical help with tissue preparation.

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