Introduction
The neuropathology of Alzheimer’s disease (AD) is characterised by the formation of extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFT) [5,34]. The most important molecular constituent of the SP is β-amyloid (Aβ) [28], an approximately 4 kDa peptide arising by constitutive cleavage of a trans-membrane amyloid precursor protein (APP). A variety of Aβ peptides are formed as a result of secretase cleavage of APP [40]. The most common of these peptides is Aβ42, found largely in discrete Aβ deposits, whereas the more soluble Aβ40 is also found in association with blood vessels [37] and may develop later in the disease [21]. The discovery of Aβ led to the formulation of the ‘amyloid cascade hypothesis’ (ACH), one of the most important models of the molecular pathology of AD developed over the last 25 years [28]. Essentially, the ACH proposes that the deposition of Aβ is the initial pathological event in AD leading to the formation of NFT, cell death, and cortical degeneration [28].
At least four genetic loci are associated with AD: the APP gene on chromosome 21 [17,26], the presenilin PSEN genes on chromosome 14 (PSEN1) [47] and chromosome 1 (PSEN2) [32], and the apolipoprotein E (Apo E) gene on chromosome 19 [44]. APP and PSEN mutation may alter APP metabolism, resulting in increased deposition of Aβ peptide, while allelic polymorphism of Apo E, and especially the expression of allele ε4, may influence the proportion of the more fibrillogenic Aβ42 formed in the tissue [32,47]. These genetic factors, however, may not explain the majority of AD cases [27]. Hence, early-onset cases linked to APP and PSEN mutations may account for less than 5% of total AD [29]. Additional susceptibility genes and environmental factors are therefore likely to be involved, especially in sporadic AD (SAD) [38,39,41]. In isolated Amish communities, for example, 24 markers have been linked to dementia [33] and several other linkage studies have shown the presence of AD susceptibility genes on chromosomes 9, 10, and 12 [48]. Hence, a small number of AD cases have been linked recently to the chromosome 9 open reading frame 72 (C9ORF72) gene [55].
Three morphological subtypes of Aβ deposit are commonly observed in AD: 1) diffuse (‘pre-amyloid’) deposits, in which the Aβ is not in a fibrillar form with a β-pleated conformation, dystrophic neurites (DN) and paired helical filaments (PHF) being largely absent, 2) primitive (‘neuritic’) deposits, in which the Aβ is in a fibrillar form and is associated with DN and PHF, and 3) classic (‘dense-cored’) deposits, in which Aβ is highly aggregated to form a central amyloid plaque ‘core’ surrounded by a ‘ring’ of DN [3,8,10-12,20]. In the cerebral cortex in AD, Aβ deposits [2] and NFT [54] often exhibit significant variation in density across the cortex from pia mater to white matter, maximum density occurring within different layers [2,54]. The laminar distribution of Aβ deposits may be a consequence of degeneration of neural pathways that have their neurons of origin or axon terminals located within particular layers [22]. The main objective of this study was to determine whether genetic factors influence the laminar distribution of Aβ deposits and therefore result in a specific type of cortical degeneration in the frontal cortex in AD. Hence, laminar distributions of diffuse, primitive, and classic Aβ deposits were studied in three groups of cases: 1) early-onset familial Alzheimer’s disease (EO-FAD) linked to mutations of either amyloid precursor protein (APP717) or presenilin 1 (PSEN1: G209V, E280A) genes, 2) late-onset familial AD (LO-FAD), and 3) sporadic AD (SAD). In addition, the influence of Apo E genotype on the distribution of Aβ deposits was studied.
Material and methods
Cases
Alzheimer’s disease cases (N = 20; details in Table I) were obtained from the Brain Bank, Department of Neuropathology, Institute of Psychiatry, King’s College, London, UK. 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 [34] and ‘National Institute on Aging (NIA)-Reagan Institute’ criteria [30,34]. The cases were divided into three groups: 1) EO-FAD (onset ≤ 65 years) (n = 4), 2) LO-FAD (≥ 65 years) (n = 6), and 3) SAD (n = 10) with no evidence of familial involvement.
Tissue preparation
A block of the frontal cortex was taken at the level of the genu of the corpus callosum to study the superior frontal gyrus (SFG). Tissue was fixed in 10% phosphate-buffered formal saline and embedded in paraffin wax. Both immunostaining and thioflavin S have been used to stain SP in AD [14,45]. Thioflavin S staining indicates that amyloid in these plaques contains fibrillar material with a β-pleated sheet conformation [14]. By contrast, immunohistochemistry generally reveals more plaques including diffuse Aβ deposits which are mainly thioflavin S-negative [14]. Hence, to label all types of plaque 7 µm coronal sections were immunolabelled 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 Aβ protein and first used to identify Aβ deposit subtypes in Down’s syndrome (DS) [50] but which also effectively distinguishes the major types of Aβ deposit in AD [4,6,7,10,50]. The antibody was used at a dilution of 1 in 1200 and the sections incubated at 4°C overnight. Sections were pretreated with 98% formic acid for 6 minutes, which enhances Aβ immunoreactivity. Aβ was visualised using the streptavidin-biotin horseradish peroxidase procedure with diaminobenzidine as the chromogen. Sections were also stained with haematoxylin. Aβ deposits were identified in the sections using criteria published by Delaere et al. [20]: 1) diffuse deposits were 10-200 µm in diameter, lightly stained, irregular in shape, and with diffuse boundaries, 2) primitive deposits were 20-60 mm, well demarcated, symmetrical in shape, and strongly stained, and (3) classic deposits were 20-100 mm and had a distinct central amyloid core surrounded by a ‘corona’ of DN [20].
Morphometric methods
The distribution of the Aβ deposits in the SFG of each case was studied from the pia mater to white matter using methods described previously [23]. Five traverses from the pia mater to the edge of the white matter were located at random within each gyrus [9]. All deposits were then counted in 200 x 1000 µm sample fields arranged contiguously, the larger dimension of the field parallel with the surface of the pia mater. An eye-piece micrometer was used as the sample field and was moved down each traverse one step at a time from the pia mater to the edge of the white matter. Histological features of the section were used to correctly position the field. The mean of the counts from the five traverses was calculated to study variations in density of histological features across each cortical gyrus.
Data analysis
No attempt was made to locate precisely the boundaries between individual cortical layers. First, the degree of cortical degeneration present in many gyri made laminar identification difficult. Second, identification was especially difficult in the frontal cortex because it exhibits a heterotypical structure, i.e., six layers cannot always be clearly identified and vary in prominence from case to case. Third, Aβ deposits appeared to exhibit complex patterns of distribution across the cortex rather than being confined to specific layers. Hence, variations in density of Aβ deposits with distance across the cortex were analysed using a polynomial curve-fitting procedure (STATISTICA software, StatSoft Inc., 2300 East 14th St, Tulsa, OK, 74104, USA) [2,49]. For each gyrus, polynomials of order 1, 2, 3 up to the 4th order were fitted successively to the data. Hence, second-order curves are parabolic, third-order curves are ‘S’ shaped, and fourth-order curves are double-peaked (bimodal). With each fitted polynomial, the correlation coefficients (Pearson’s ‘r’), regression coefficients, standard errors of the mean (SEM), values of t, and the residual mean square were obtained. At each stage, the reduction in the sums of squares (SS) was tested for significance. A polynomial was accepted as the best fit using the procedure described by Snedecor and Cochran [49], viz. when either a non-significant value of F was obtained or there was little gain in explained variance. The distributions of the Aβ deposits across the cortex were classified initially into three groups: 1) a single density peak was present (unimodal distribution), peak density being located in either upper or lower layers, 2) two density peaks were present (bimodal distribution), density peaks occurring in upper and lower layers, and 3) there was no significant change in density across the cortex, Aβ deposits not being confined to particular layers. Bimodal distributions were then classified further according to whether the density peaks in the upper and lower layers were of similar or different magnitude. To study the effect of Apo E genotype, cases were classified into two groups: those not expressing allele ε4, i.e., genotypes ε2/3 and ε3/3, and those expressing at least one allele ε4, i.e., genotypes ε3/4 and ε4/4. In addition, the location of peak density and the maximum density of deposits were compared in EO-FAD, LO-FAD, and SAD. Hence, the point of maximum density (peak density) was identified for each deposit type for each gyrus while location of the peak was determined as the distance from the pia mater to that of the maximum density of Aβ deposits, expressed as a percentage of the total distance from the pia mater to the edge of the white matter.
Results
Examples of the distribution of Aβ peptide deposits across the SFG are shown in Figures 1 and 2. In a case of EO-FAD (Fig. 1) (PSEN1 mutation), Aβ deposits occurred across the cortex with a greater density of larger deposits in the upper layers. By contrast, in a case of SAD (Fig. 2), Aβ deposits occur largely in the lower layers.
Examples of the laminar distribution of the diffuse, primitive, and classic Aβ deposits in the SFG of a single EO-FAD case (case 2, APP mutation) is shown in Figure 3. The distribution of the diffuse deposits was fitted by a first-order (linear) regression (r = 0.82, p < 0.01) consistent with greater densities of diffuse deposits in the upper layers and a linear decrease in density across the cortex from pia mater to white matter. The distribution of the primitive Aβ deposits was fitted by a third-order polynomial (r = 0.91, p < 0.001) with a large density peak in the upper layers, while the classic deposits were also fitted by a third-order polynomial (r = 0.82, p < 0.01) with slightly higher densities adjacent to the pia mater and in the lower layers.
A comparison of the laminar distributions is shown in Table II. In FAD, diffuse Aβ deposits exhibited a density peak in the upper layers in 6/10 cases, and the primitive deposits did so in 9/10 cases. The distribution of the classic deposits was more variable; in 6/10 cases there was either a density peak in lower cortex or a bimodal distribution was present with density peaks in upper and lower layers. In SAD, diffuse and primitive Aβ deposits exhibited a density peak in the upper cortex in 7/10 cases and 9/10 cases respectively. Distribution of the classic Aβ deposits was more variable, a density peak in the lower layers or a bimodal distribution being present in 5/10 cases. The frequency of the various types of distribution of the diffuse (2 = 3.74, p > 0.05), primitive (2 = 0.71, p > 0.05), and classic (2 = 11.18, p > 0.05) Aβ deposits was similar in EO-FAD, LO-SAD, and SAD.
Comparison of the mean location of maximum density and peak density of deposits among the three groups of cases is shown in Table III. Although there were significant differences in the layers at which peak density occurred among Aβ deposit subtypes (F = 4.44, p < 0.01), there were no significant differences among EO-FAD, LO-FAD, or SAD (F = 0.89, p > 0.05). In addition, there were no significant differences in peak density of Aβ deposits (F = 3.28, p > 0.05) among patient groups.
A comparison of the distributions exhibited by the Aβ deposits in cases classified according to Apo E genotype groups is shown in Table IV. There were no significant differences in distribution of diffuse (2 = 2.55, p < 0.05), primitive (2 = 0.003, p < 0.05), or classic (2 = 3.41, p < 0.05) Aβ deposits in cases expressing Apo E genotypes ε2/3 and ε3/3, compared with those expressing genotypes ε3/4 and ε4/4.
Discussion
The objective of this study was to determine whether genetic factors were associated with a specific pattern of cortical degeneration, as revealed by the deposition of Aβ deposits in the frontal cortex in AD. The data confirm the need for quantitative assessment of Aβ deposition in different layers of cortex as deposits often occur over many layers with variation in abundance across the cortex. This study demonstrated: 1) laminar distributions of diffuse, primitive, and classic Aβ deposit subtypes were essentially similar in EO-FAD, LO-FAD, and SAD, 2) within FAD, laminar distributions were similar in APP/PSEN1 cases compared with LO-FAD, and 3) laminar distributions were similar in cases expressing Apo E ε4 alleles compared with cases expressing ε2 or ε3 alleles.
The data suggest no significant differences in Aβ deposit density in EO-FAD, LO-FAD, and SAD or when cases were classified according to Apo E genotype. Previous quantitative studies comparing SP or Aβ deposit abundance in FAD and SAD have been controversial [16,18,24,36]. Hence, no significant differences in severity scores of SP were observed in FAD and SAD [36], and Aβ ‘load’ in the frontal cortex and temporal isocortex was similar in SAD and FAD cases linked to the APP717 mutation [16]. Nevertheless, cultured cells expressing a double mutation in APP produced six times more Aβ than normal cells [18]. In addition, other studies have reported increased amyloid deposition in individuals expressing allele ε4 [24]. However, it is possible that Aβ deposition could be more widely distributed across the cortical layers of the SFG in FAD, but with similar peak densities.
In the SFG of both SAD and FAD, maximum density of the diffuse and primitive Aβ deposits occurred most frequently in the upper layers. By contrast, the distribution of the classic deposits was more variable, peak densities occurring either in the lower layers, or in both upper and lower layers. Similar results have been reported in studies of the laminar distribution of SP [15,19], Apo E-immunoreactive SP [53], neuritic plaques (NP) [42], and Aβ deposits in AD [2], which are frequently abundant in layers II and III. In addition, in a transgenic mouse model expressing the APP717 mutation, Aβ deposits were most abundant in layers II and III, similar to AD [51]. However, aged dogs often show a different distribution of Aβ deposits to humans, being usually abundant in the deep cortical layers but with evidence of spread to superficial cortical layers with increasing age [43].
Various hypotheses could explain the laminar distribution of Aβ deposits in the SFG in AD. First, mRNA of APP is preferentially expressed by the large pyramidal neurons in layers III and V [13]. Degeneration of these neurons could then result in increased secretion of APP and formation of Aβ deposits within these layers [4]. Second, interleukin-immunoreactive microglia (IL-Mg) have a similar laminar distribution as APP-immunoreactive NP [46]. Hence, the laminar distribution of microglia could be a factor determining the distribution of the Aβ deposits. Third, the laminar distribution of the classic deposits could be spatially related to blood vessels [2,37]. Large blood vessels often exhibit a bimodal distribution in the cortex, whereas smaller capillaries occur at maximum density in the deeper layers [2]. In addition, Akiyama et al. [1] found that Aβ deposits accumulated vertically in columns, with blood vessels often occurring perpendicular to the column and penetrating its centre. Previous studies suggest, however, that although classic Aβ deposits are clustered around blood vessels in SAD [6], there are fewer spatial associations with blood vessels in FAD [7].
Laminar distributions of Aβ deposits in frontal lobe AD are essentially similar in the FAD and SAD cases examined and similar whether Apo E allele ε4 was present or not [31]. In addition, among FAD cases, there was no evidence that a specific type of laminar distribution was influenced by genetic subtype. Hence, neither APP/PSEN1 mutations nor the presence of Apo E allele ε4 uniquely determines Aβ deposition and therefore the pattern of frontal lobe degeneration in AD. Uchihara et al. [53] found that Apo E labelled a subset of deposits in lamina III with more Apo E-immunoreactive diffuse deposits in the deeper layers. However, only a proportion of the diffuse deposits were Apo E-immunoreactive, suggesting that Apo E was not involved in the process of cortical degeneration but immunoreactivity was acquired by certain deposits later in the disease. Hence, pathological changes initiated by the various genetic changes in FAD and, by other causes in SAD, appear to follow a parallel course resulting in very similar patterns of cortical degeneration in the SFG.
In conclusion, there were no essential differences in the laminar distribution of the Aβ deposits in the SFG between FAD and SAD, or between different subtypes of FAD. Hence, APP and PSEN1 mutations and the presence of Apo E genotype ε4 appear to have little influence on laminar distribution. Although the mechanism of generating fibrillogenic species of Aβ may differ among disease subtypes, gene expression appears to have little effect on the pattern of degeneration of the frontal lobe in AD.
Acknowledgments
Brain tissue sections for this study were kindly provided by the Brain Bank, Department of Neuropathology, Institute of Psychiatry, King’s College London, UK. Mrs Mavis Kibble and Mr Alan Brady are thanked for technical help with tissue preparation.
Disclosure
Author reports no conflict of interest.
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