Introduction
There are similarities in the pathology of Alzheimer’s disease (AD) and Creutzfeldt-Jakob disease (CJD) [7,20]. Both disorders are characterized by the deposition of a protease resistant protein aggregate in the form of deposits or plaques, viz., β-amyloid (Aβ) in AD and the disease associated form of prion protein (PrPsc) in CJD. In some patients, Aβ and prion pathology can even coexist [20,32,34], with in one reported family the features of both CJD and AD pathology linked to a presenilin 1 (PSEN1) mutation [19]. In addition, Aβ and PrPsc deposits exhibit similarities in spatial topography, being distributed in clusters that in the cerebral cortex are regularly distributed parallel to the pia mater [7]. Aβ is generated via β and γ-secretase cleaving of amyloid precursor protein (APP), while PrPsc is an abnormal structural conformation of normal cellular PrP (PrPc) and results from the autocatalytic conversion of PrPc to PrPsc. The formation of protein aggregates is a nucleated polymerization reaction with an initial nucleation event (lag phase) followed by the extension of newly formed nuclei into larger aggregates (growth phase) [16,21,29]. Several studies have suggested that Aβ deposits grow in the brain. Pseudocolour image processing reveals gradients of density within Aβ deposits consistent with growth from a central point [13]. Radioiodinated human Aβ can be deposited experimentally in vitro from a dilute solution onto primitive and diffuse deposits, causing them to grow [30]. In transgenic mice, Aβ deposits appear in clusters which grow in size from 14 µm at 8 months to 22 µm at 12 months [41]. Transgenic studies also suggest that increasing accumulation of Aβ is largely by growth of existing deposits rather than by further nucleation [36]. The size frequency distributions of protein deposits in thin sections of tissue have been used to investigate the growth phase [3,23,38]. Hence, in AD and Down’s syndrome (DS), Aβ deposits exhibit a unimodal, positively skewed distribution [6,23]. There were few deposits in the smallest size class (plaque diameter <10 µm), maximum frequency occurred between 20 µm and 40 µm (the modal class), and the frequency of the larger deposits declined with increasing size. A log-normal model has been fitted to the size frequency distributions of Aβ deposits in DS [23,38]. This model suggests that after nucleation, the growth phase of the Aβ deposits can be described by a function in which increase in volume of a deposit in a time interval is proportional to its volume at the beginning of the time interval (dV/dt = K(t)V, where V = deposit volume and K(t) is a parameter changing randomly around a constant positive value) [23]. Previous studies also suggest that PrPsc deposits in CJD exhibit a size distribution similar to that of Aβ deposits in AD [12]. Hence, the present study compared the size frequency distributions in the temporal lobe of Aβ deposits in AD with deposits of PrPsc in the variant subtype of CJD (vCJD) [8]. Variant CJD is a relatively new form of the disease first described in the UK in 1996 [43] and has protein deposits that morphologically resemble those of AD [8].
Materials and methods
Cases
Ten cases of sporadic AD (Table I) were obtained from the Brain Bank, Department of Neuropathology, Institute of Psychiatry, King’s College London, UK, and 11 vCJD cases from the National CJD Surveillance Unit, Western General Hospital, Edinburgh, UK. Informed consent was given for the removal of all brain tissue according to the 1996 Declaration of Helsinki (as modified Edinburgh 2000). The AD cases were clinically assessed and all fulfilled the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer’s Disease and Related Disorders Association (NINCDS/ADRDA) criteria for probable AD [39]. The histological diagnosis of AD was established by the presence of widespread neocortical senile plaques (SP) consistent with the Consortium to Establish a Registry of Alzheimer’s Disease (CERAD) criteria [31]. AD cases conformed to stages IV to VI of the Braak system [14]. All CJD cases fulfilled the neuropathological diagnostic criteria for vCJD [24]. None of the cases had any of the known mutations of the PrP gene or family history of prion disease, and there was no evidence of the known types of iatrogenic aetiology. The PrPsc characteristic of vCJD has a uniform glycotype (PrPsc, Type 4) and is distinct from that observed in sporadic CJD [22,24].
Histological methods
A block of the temporal cortex was taken from each case at the level of the lateral geniculate nucleus to study the superior temporal gyrus (B22), inferior temporal gyrus (B20), and parahippocampal gyrus (B28). Tissue was fixed in 10% phosphate buffered formal-saline and embedded in paraffin wax. In AD, coronal 7 µm sections were stained with a rabbit polyclonal antibody (provided by Professor B.H. Anderton, Institute of Psychiatry, King’s College London) raised against the 12-28 amino acid sequence of the Aβ protein (dilution 1:1200) [37]. In vCJD, coronal 7 µm sections were immunostained against PrPsc using the monoclonal antibody 12F10 (dilution 1:250) that binds to residues 142-160 of human PrP downstream of the neurotoxic domain adjacent to helix region 2 [27] (provided by Prof. G. Hunsmann, The German Primate Centre, Gottingen, Germany). Immunoreactivity was enhanced by formic acid (98% for 5 minutes) and autoclaving (121°C for 10 minutes) pretreatment. Sections were treated with Dako Biotinylated Rabbit anti-Mouse (RAM) (dilution 1:100) and Dako ABComplex HRP kit for 45 minutes (Amersham, UK). Diaminobenzidine tetrahydrochloride was used as the chromogen. All sections were counterstained with haematoxylin for 1 minute.
Morphometric methods
Protein deposits in AD and vCJD have a similar morphology [2,9]. In both disorders, there are diffuse deposits (also known in vCJD as ‘fine feathery diffuse deposits’ or ‘fine diffuse plaques’) and more complex deposits in which a distinct central ‘core’ of Aβ or PrPsc is present and are termed ‘classical’ plaques in AD or ‘florid’ plaques in vCJD [9]. Within each gyrus, the greatest diameter of a sample of diffuse and classic/florid deposits was measured. Guidelines were marked on the slide parallel to the pia mater in laminae II/III and V/VI, regions that contain the most significant numbers of deposits [10]. The maximum diameter of each protein deposit touching a guideline was then measured at a magnification of ×400 using an eyepiece micrometer. Diffuse Aβ deposits were 10-200 µm in diameter, irregular in shape with diffuse boundaries and lightly stained, while classic deposits had a distinct central core surrounded by
a ‘corona’ of dystrophic neurites [2]. Florid PrPsc deposits were unicentric and consisted of a dense core, while diffuse deposits were irregularly shaped, more lightly stained than the florid deposits and always lacked a solid core [9]. To obtain a sufficiently large sample of each type of deposit within each disease group to fit a log-normal model, measurements were combined from all brain regions and patients.
Data analysis
A log-normal model was fitted to the size distribution of each type of deposit [35] using STATISTICA software (Statsoft Inc., 2300 East 14th St, Tulsa, OK 74104, USA). The log-normal distribution is defined as that of a variable X such that ln (X-Ø) is normally distributed. The distribution has three parameters: Ø (where X > Ø), the mean (µ), and the variance (σ2). In many applications, the value of Ø can be assumed to be zero and a two-parameter model fitted to the data. Deviations from a log-normal model were tested using the Kolmogorov-Smirnov (KS) goodness-of-fit test.
Results
The size frequency distributions of the Aβ deposits are shown in Figures 1 and 2 and the PrPsc deposits in Figures 3 and 4. The mean size, modal class, standard deviation, maximum size and degree of skew of the distributions are summarised in Table II. All size distributions were unimodal and positively skewed. There were few deposits represented in the smallest size classes, maximum frequency occurred at upper boundary sizes of 30-50 µm (the modal class) for the Aβ deposits and 5-25 µm for the PrPsc deposits, while the frequency of the larger deposits declined rapidly with increasing size. Average and maximum diameter of the diffuse Aβ deposits was greater than that of the corresponding diffuse PrPsc deposits, and similarly, the classic Aβ deposits were larger than the florid PrPsc deposits. The PrPsc deposits had smaller standard deviations but exhibited a greater degree of skew than the Aβ deposits. All distributions were approximately log-normal in shape. However, with the exception of the diffuse deposits in vCJD (Fig. 3), the distributions showed some deviations from the expected numbers of deposits predicted by the log-normal model. Hence, there were more diffuse Aβ deposits (Fig. 1) with an upper size limit of <50 µm in diameter and fewer >50 µm than expected and there were more classic Aβ deposits (Fig. 2) <20 µm and fewer between 20 µm and 40 µm than expected, while the numbers of deposits >60 mm were closer to those predicted by the log-normal model. There were fewer florid PrPsc deposits (Fig. 4) <4.5 µm, more in the modal class (5 µm), and fewer >18 µm than predicted by the log-normal model.
Discussion
Both Aβ and PrPsc deposits exhibited a unimodal, positively skewed size distribution, suggesting similarities in the growth phase of both types of deposit. The average and maximum size of the Aβ deposits, however, was significantly greater than the analogous PrPsc deposits. Either Aβ deposits increase in size more rapidly or develop over a much longer time period than PrPsc deposits. Although vCJD cases have a duration of up to two years, considerably longer than in sporadic CJD [43], duration of disease is still much shorter than in AD. However, in the AD cases with a short duration (2-3 yrs), the sizes of the Aβ deposits were still significantly greater than those of the PrPsc deposits, suggesting that Aβ deposits have an intrinsically greater growth potential. In addition, the PrPsc deposits exhibit a greater degree of skew and smaller standard deviations than the Aβ deposits. This suggests that the pattern of growth of the PrPsc deposits is less variable than the Aβ deposits and that there are factors that restrict the growth of the PrPsc deposits to within a certain size range. Development of protein deposits is characterised by two processes, viz., growth and removal of protein molecules (aggregation/disaggregation) and the diffusion of substances into the deposit (surface diffusion) [38,41]. The size frequency distribution approaches log-normal if surface diffusion predominates over that of aggregation/disaggregation. In a previous study [12], the distributions of the diffuse and the florid PrPsc deposits deviated significantly from a log-normal model. The present data, comprising a large sample of deposits from the temporal lobe, differ in that the diffuse PrPsc deposits did not deviate significantly from a log-normal distribution. Hence, surface diffusion may be important in the development of the diffuse deposits. Diffuse deposits may acquire small molecular weight ligand substances readily, the substances contributing to growth by binding to PrPsc and promoting the formation of peptide bonds [28]. For example, clusterin is a heterodimeric glycoprotein which has a propensity to form aggregates and which can interact with PrPsc [17]. Compliment activation products C1q and C3d, serum amyloid P, and activated glial cells may also accumulate in prion deposits and influence their growth and development [42]. These substances are also found in diffuse Aβ deposits [4] but may have little effect on the growth phase. The remaining size distributions deviated from a log-normal model. Most notably, there were far fewer florid PrPsc deposits in the smallest size class (<4.5 µm) than predicted. Sampling in two dimensions underestimates the frequency of small deposits [6,25] and this sampling error may be particularly significant in measuring the small florid deposits. Alternatively, fibril formation is nucleation dependent and occurs after a lag time which decreases with increasing peptide concentration [33]. Once small amorphous aggregates are formed and β-sheet formation is initiated there is rapid growth of the stable fibrils or protofibrils to form the deposits [33]. Hence, rapid early growth of the florid deposits could also explain the low numbers of small deposits observed. There were fewer of the large diffuse Aβ and florid PrPsc deposits than predicted by the log-normal model, consistent with the suggestion that there is an upper limit to the growth of these deposits. During the growth phase of Aβ deposits, new amyloid fibres are formed at the periphery of existing deposits [44] with specific amyloid aggregate formation accelerated by the homogeneous association of soluble Aβ42 onto existing Aβ42 seeds [18]. By contrast, growth of a PrPsc deposit is dependent on the continued autocatalytic conversion of PrPc. Hence, growth of a deposit will depend on a supply of the precursors of Aβ and PrPsc, viz., amyloid precursor protein (APP) and PrPc respectively, both of which are neuronal proteins of uncertain function. The size of an Aβ deposit is positively correlated with the number of associated neuronal perikarya [6]. Hence, deposit size may be restricted by the number of immediately adjacent neurons that degenerate and secrete the proteins necessary to form the deposit (Armstrong et al., 1997). Furthermore, both the classic deposits in AD [1,5] and the florid deposits in vCJD [11] have been observed to cluster around the vertically penetrating arterioles in the upper laminae of the cerebral cortex, suggesting that factors associated with blood vessels or blood are important. Substances diffusing from blood into the brain as a result of an impaired blood brain barrier might encourage condensation of the protein to form a dense core, thus restricting the size of the deposit [11]. The role of aggregated proteins in the pathogenesis of AD and CJD is controversial. The aggregates themselves may be toxic and therefore these results may be useful in the design of treatments that may act to restrict their growth and spread. Alternatively, in AD, Aβ oligomer intermediates may be the toxic species [26] and amyloid formation and deposit growth could represent a protective mechanism that actually removes the toxic species from the brain [15]. In this case, studies of the size frequency distributions of protein deposits may be a useful means of studying this potentially important protective mechanism.
Acknowledgements
The assistance of the Brain Bank, Institute of Psychiatry, King’s College London in preparation of tissue sections for this study is gratefully acknowledged. The CJD Surveillance Unit is supported by the Department of Health, the Scottish Executive and TSELAB Project of the EC (reference QLK2-CT-2002-81523).
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