See also: 2.0, 3.1, 5.1, 8.3, 8.6

1.0 Amyloid beta-protein (Abeta) positive granules in astrocytes, microglia and brain macrophages. H. Akiyama(1), H. Kondo(1), H. Mori(1), K. Ikeda(1), and P. L. McGeer(2). (1)Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156, Japan, (2)Kinsmen Laboratory, University of British Columbia,Vancouver, B.C., V6T 1Z3, Canada. E-mail: akiyama@prit.go.jp

Keywords: microglia, astrocyte, phagocytosis, removal

Abstract: In postmortem brain tissues from patients with Alzheimer's disease (AD), granules immunopositive for amyloid beta-protein (Abeta) are present in a subset of microglia and astrocytes. Such Abeta containing glial cells occur in areas with the extracellular Abeta deposits. In this study, an array of antibodies to Abeta as well as antibodies to some molecules associated with activation of microglia or astrocytes were used to characterize the Abeta positive granules and glial cells that contain such granules. These include Abeta1 (end-specific to N-terminal Asp(1)), 6E10 (A beta5-8), 6F/3D (Abeta11-15), 4G8 (Abeta18-20), E50 (Abeta22-31), Abeta40 (end-specific to C-terminal Val(40)), and Abeta42 (end-specific to C-terminal Ala(42)/Thr(43)), as well as antibodies to glial fibrillary acidic protein, CD44, intercellular adhesion molecule-1, type I IgG Fc receptor, complement receptor CR4, CD68, CD45RO, and HLA-DR.

Abeta in glial granules is positive for 4G8 and E50, but negative for Abeta1, 6E10, and 6F/3D. Absence of epitopes recognized by antibodies to the N-terminal fragments suggests the truncation around alpha-secretase cleavage site. The exact N-terminus of Abeta in glial granules remains to be determined, however.

Occurrence of Abeta containing glial cells varies greatly from patient to patient and from area to area in a given patient. In many patients, such glial cells are present in the subiculum. Abeta42 predominates Abeta40 in the majority of extracellular Abeta deposits in the subiculum. Abeta in glial granules associated with such deposits is Abeta42 positive but Abeta40 negative.

Occasionally, Abeta containing glial cells appear in association with a peculiar type of diffuse Abeta deposits. This type of Abeta deposits consist of lightly stained amorphous materials. Both Abeta40 and Abeta42 are present but, in most instances, Abeta40 predominates Abeta42 in these deposits. Intraglial Abeta granules associated with these deposits contain both Abeta40 and Abeta42. The Abeta40 positive diffuse deposits with many glial granules occur in the neocortex of AD patients but only inconsistently. In the vast majority of tissue sections examined in this study, most Abeta deposits were not of this type. These deposits often formed clusters in areas of up to several hundred micrometers in diameter. Expression of activation-related molecules by glial cells was not upregulated in such clusters.

A few AD patients investigated in this study were complicated with ischemic brain lesions. In areas that have been subjected to recent ischemia, reactive microglia and macrophages contain Abeta positive granules. Staining of the extracellular Abeta deposits is much less intense than in the non-ischemic surround, indicating removal of the Abeta deposits by these phagocytic cells. Immunohistochemical profile of Abeta granules in macrophages is similar to that of intraglial granules. Such results suggest that digestion of the N-terminal sequences is an early consequence of this phagocytosis.

Taken together, we speculate that extracellularly released or deposited Abeta is taken up by microglia and astrocytes. In some conditions, Abeta accumulates in these glial cells and becomes visible in postmortem tissue sections.

1.1 Amyloid precursor protein expression and intracellular accumulation of amyloid beta peptide during differentiation of human neuroblastoma IMR-32 cells. I.V. Kurochkin (1), H.H. Ho (2), S. Ikeda (3), C. Schönbach (1). (1) Chugai Research Institute for Molecular Medicine, Inc. Ibaraki 300-41, Japan, (2) Choongwae Pharma Corporation, Kyungkido, Republic of Korea, (3) Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka 412, Japan. E-mail: igork@tk.chugai-pharm.co.jp

Keywords: Cellular and Animal Models, Alzheimer's disease, Amyloid beta peptide, Differentiation, IMR-32 cell, Serotonin neuron

Abstract: The cerebral deposition of amyloid beta peptide (Abeta) and development of neurofibrillary tangles (NFT) are invariant features of Alzheimer's disease (AD). The relationship between these two lesion types in AD is not established, although it is believed that deposition of Abeta precedes formation of NTF. Recently reported observation that intracellular Abeta accumulation precedes extracellular Abeta deposition in the aged monkey brain and well-documented presence of Abeta in NFT prompts speculation that intracellular Abeta may be involved in NFT formation. In order to investigate this issue, a model cell line displaying both pathological phenomena seen in AD is required. Of several human neuroblastoma cell lines so far tested, only IMR-32 cells when exposed to the differentiation medium develop intracellular fibrillary material that reacts with AD specific NTF antibody. The main aim of the present study was to investigate whether neuronaly differentiated IMR-32 cells produce intracellular Abeta and if so establish temporal and quantitative relationship between amyloid precursor protein (APP) expression and intracellular Abeta levels during differentiation.

BrdU, 5-bromodeoxyuridine, added to the medium induced marked morphological changes toward a neuronal phenotype with concomitant increase in tyrosine hydroxylase (TH) expression. The APP levels, barely detectable before the induction, increased rapidly after BrdU treatment rising 200-fold between days 1 and 15. Temporal pattern of APP expression was very close to that of TH. Intracellular Abeta was analyzed by immunoprecipitation of cell extracts with the anti-Abeta antibody after metabolic labeling IMR-32 cells with [35S] methionine. Unlike APP, Abeta could be detected only at later stage of differentiation. The levels of cell-associated Abeta increased more than tenfold between days 5 and 9, while APP increased only about twofold within the same period of time. Using antibody raised against C-terminal part of APP we also could detect in the cell extracts the 12 kDa APP fragment representing likely APP amyloidogenic intermediate. Again, as in case of Abeta, the 12 kDa fragment appeared late in differentiation, when the APP levels have almost reached its maximum.

The data obtained support the concept that APP plays an important role in neuronal development. Temporal relationship between the APP expression and intracellular Abeta accumulation indicates that APP metabolism is altered toward amyloidogenic pathway at late stages of neuronal differentiation. Since differentiated IMR-32 cells is the only human cell line known to develop NFT, it can be a suitable model system to study relationship between accumulation of intracellular Abeta and formation of the pathological neuronal fibrillary structures. In addition, this study suggests that the serotonergic neurons, what differentiated IMR-32 cells are, may contribute to amyloidogenesis in AD.

1.2 Signal transduction mechanisms associated with amyloid precursor protein secretion from rat presynaptic terminals. M. McLaughlin & K.C. Breen. Neurosciences Institute, Dept. of Pharmacology, University of Dundee, Ninewells Hospital Medical School, Dundee DD1 9SY, Scotland, U.K. E-mail: m.mclaughlin@dundee.ac.uk

Keywords: Amyloid, Kinase, Synaptosome, Secretase, Processing

Abstract: One of the hallmarks of Alzheimer's disease is the deposition of senile plaques which are composed primarily of the beta A4 protein. Studies of the mechanisms associated with beta A4 generation have established that its production is decreased by the activation of protein kinase C (PKC) with a concomitant increase in the secretion of soluble APPs. However such investigations are mainly conducted using transfected sytems and may not accurately represent the situation in vivo. We have employed a synaptosome preparation to examine the signaling processes associated with APPs secretion since this model can permit the integration of biocheical studies with the functional analysis of neurotransmitter release and calcium influx. We have establihsed, using a range of specific antisera, that APP is present in both cortical and hippocampal preparations and is predominantley localised to the membrane. APPs secretion occurs in a time depedent manner suggesting a constitutive secretese activity. The secretion is also stimulated by the activation of PKC and this enhancement is blocked by preincubation by PKC inhibitors. Thus, rat synaptosomes are an ideal model for examining the mechanisms associated with APP processing at the presynaptic level and should prove valuable in establishing the role of APP in neuronal excitability.

1.3 Copper-Mediated Aggregation and Polymerization of Alzheimer Aß is Promoted By Conditions Representing Physiological Acidosis. C.S. Atwood, X. Huang, R.D. Moir, R.C. Scarpa, N.M.E. Bacarra, M.A. Hartshorn, L.E. Goldstein, D.M. Romano, R.E. Tanzi and A.I. Bush. Genetics & Aging Unit, Departments of Psychiatry and Neurology, Massachusetts General Hospital, Boston, MA, 02129. E-mail: atwoodc@helix.mgh.harvard.edu

Keywords: Amyloid beta, Copper, SDS-resistant polymerization, Free radicals, Aggregation

Abstract: The cortical deposition of Aß, a normally soluble and constitutive 4.3 kDa peptide of the CNS and periphery, is observed in Alzheimer's disease, Down's syndrome, head injury and normal aging. Previously we found that Zn(II) induces the aggregation of soluble Aß in vitro. Using sedimentation and turbidometric assays to further quantitate the effects of neurochemical factors on Aß solubility, we now report that unlike other biometals, marked Cu(II)-induced aggregation of Aß1-40 occurs as the solution pH is lowered from 7.4 to 6.8. This rapid, pH-sensitive aggregation of Aß1-40 was completely reversible by alkalinization. These results indicate that H+-induced conformational changes unmask a metal binding site on the peptide that modulates its behavior in response to changing pH. Since a mildly acidic environment, together with increased Zn(II) and Cu(II) are common features of inflammation, we propose that Aß1-40 aggregation by these factors could be a physiological, and reversible, response to neuronal injury.

The modification of Aß into SDS-resistant (SR) polymers is a defining neuropathological feature of AD. To test whether redox active metal ions could promote SR polymerization of Aß, Aß1-40 was incubated with Cu(II). Western blot analysis revealed that Cu(II) induced the formation of SR polymers of Aß1-40 over days. In contrast, Cu(II)-induced SR polymers of Aß1-42 were detected within minutes. Cu(II)-induced SR polymerization of Aß1-42 was potentiated by mildly acidic conditions, but SR polymerization was not observed at any pH with Fe(III) or Zn(II). Exogenous H2O2 promoted the formation of SR Aß polymers in the presence of metals in the order Cu(I)âCu(II)>>>Fe(II)>Fe(III) (no SR polymerization). These results suggested that metal ion reduction was required in the formation of SR Aß polymers.

Cu(II)-mediated SR polymerization was attenuated under anoxic conditions, and by catalase, confirming that the generation of reactive oxygen species, in particular hydrogen peroxide (H2O2), was essential for the formation of SR polymers. Since the highly reactive hydroxyl radical (OH.) is generated from H2O2 in the presence of reduced metal ions, the formation of SR polymers may be a result of OH. induced covalent modification of the peptide. Reduced metal ions are released from metalloproteins under mildly acidic conditions. Thus, lowered cerebral pH, which is a complication of aging and injury could promote the generation of reactive oxygen species and the formation of SR Aß polymers that deposit in amyloid plaques.

Supported by: NIH (1R29AG1268601), AFAR (Paul Beeson Award), Alzheimer's Association (IIRG-94110), International Life Sciences Institute.

1.4 Augmentation of calcium channel currents in cultured rat cerebellar granule neurones by amyloid beta protein. H A Pearson and S A Price. Department of Pharmacology, University of Leeds, Leeds LS2 9JT, UK. E-mail: h.a.pearson@leeds.ac.uk

Keywords: amyloid beta protein, electrophysiology, calcium channels, nifedipine, conotoxin

Abstract: Amyloid beta protein (ABP) has previously been shown to increase Ca2+ channel currents in an immortalised cell line (Davidson et al, 1994). A notable feature of the effect of ABP in these cells was a shift in the voltage dependence of Ca2+ channel activation. We have studied the effects of ABP (1-40) on the Ca2+ channel currents in cultured rat cerebellar granule neurones with a view to determining; a) the Ca2+ channel subtypes affected by ABP and b) whether other voltage-dependent gating characteristics were altered. Cultured rat cerebellar granule neurones were grown in culture, and whole-cell recordings made, as previously described (Pearson et al, 1995). Currents were activated by step depolarising cells from a holding potential of 90mV to a range of test potentials for 20ms. For incubation of cells, ABP (1-40) was added to cell culture medium to give a final concentration of 1 M and cells were left for 24 hours before recording. For controls, another group of cells from the same batch were incubated in the reverse sequence peptide (ABP, 40-1) over the same time period. Preincubation of cells with 1 M ABP(1-40) for 24 hours resulted in an increase in the mean Ca2+ channel currents when compared to controls (n=24 control cells, 26 ABP-treated cells, p<0.02, students t-test). The potential at which 50% of current was activated was shifted from -5.1+/-1.5mV in control cells to 0.5+/-1.0mV in ABP-treated cells (p<0.02). Furthermore, the rate at which Ca2+ channel currents deactivated following repolarisation was altered by ABP. The time constant of tail current decay was 2.1+/-0.4ms for controls cells but decreased to 1.1+/-0.1ms for ABP-treated cells (p<0.05). To determine which Ca2+ channel subtype's activity was augmented by 24h incubation with ABP, currents were recorded from control cells (n=14) and cells preincubated with ABP (n=15) in the presence of 2 M nifedipine. Under these conditions the majority of the L-type Ca2+ channel activity should be blocked. Nifedipine was unable to prevent the increase in Ca2+ channel activity seen following treatment of cells with ABP (p<0.05), suggesting that L-type Ca2+ channels were not involved.

In contrast to these results with the L-type antagonist, the N-type antagonist w CgTX (1 M) was able to reduce the increase in current caused by ABP (n= 13 controls, 11 ABP). No significant differences in the Ca2+ channel current were seen between controls and ABP treated cells at any of the potentials used. There was however, a consistent, but non-significant, increase in currents at potentials positive to +10 mV, suggesting that the increased current is carried only partly by N-type channels. Thus 24h preincubation of cerebellar granule neurones with ABP augments the Ca2+ channel current. This is due to an increase in N-type Ca2+ channel activity although other Ca2+ channel subtypes may also be involved. In addition, ABP shifted channel activation by +5mV and also caused an incease in the rate of channel deactivation.

Supported by the Nuffield Foundation and the Physiological Society.

References:
1) Davidson RM, Shajenko, L & Donta, TS (1994). Brain Res. 643; 324-327
2) Pearson, HA Sutton, KG, Scott, RH & Dolphin, AC (1995). J. Physiol. 482; 493-509

1.5 The interaction between chronic low-level lead and the amyloid ß precursor protein. F.D. Hayes, K.C. Breen. Dept. of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital Medical School, Dundee DD1 9SY, Scotland, U.K. E-mail: K.C.BREEN@dundee.ac.uk

Keywords: Amyloid, Lead, Toxin,: Neuroprotective, Processing

Abstract: The amyloid ss precursor protein (ABPP) plays a pivotal role in this development, both as a neurotrophic/neuroprotective factor and as a mediator of cell adhesion. In this study, we have used an in vitro system to examine the interaction between chronic low-level lead exposure and the expression and function of ABPP. Chronic exposure of the HN9 rat hippocampal cell line to lead chloride (10-14M to 10-6M) for 96 hours resulted in a 50% increase in the levels of the particulate form of the protein with a parallel decrease in the soluble form (ABPPs). This effect of lead was reversible following removal of the toxin and could be blocked by the protein synthesis inhibitor cycloheximide. This increase in membrane-bound ABPP was also paralleled by an increase in cell adhesivity to a fibronectin substrate. In addition, ABPPs also acted to attenuate lead toxicity. Cells which secreted high levels of the protein were resistant to lead toxicity when compared with control cells suggesting that the protein may be acting to chelate the metal and thus attenuating its toxic action within the cell.

1.6 Pharmacological modulation of amyloid precursor protein (APP) metabolism:biological bases and pathology-related changes. M. Racchi, L. Gasparini, M. Trabucchi and S. Govoni. IRCCS "Centro San Giovanni di Dio - Fatebenefratelli, Brescia and Institute of Pharmacology, University of Pavia, ITALY. E-mail: govonis@ipv36.unipv.it

Keywords: Amyloid precursor protein, Human fibroblasts, Protein Kinase C, Bradykinin, Oxidative metabolism

Abstract: Aberrant regulation of APP metabolism may be a causative factor in the overproduction of amyloidogenic fragments, leading to the progressive deposition of extracellular amyloid and to the development of Alzheimer's disease (AD) pathology. An action at this level may result in slowing the process of the disease and may be additive to other properties of drugs considered for AD treatment. Within this context, using skin fibroblasts from sporadic AD patients we demonstrated a reduced basal secretion of soluble APP (sAPP) from AD cells, indicating a constitutive deficiency of APP processing in spite of no difference in APP expression. Furthermore, we observed a reduced stimulated sAPP release from AD cells in response to protein kinase C (PKC) direct stimulation with nanomolar phorbol dibutyrate (PdBu). This defect appeared to be correlated with a specific defect of PKCalpha, the cytosolic concentrations of which were reduced in AD fibroblasts. Our data (Neurosci.Lett., 201, 1-4, 1995) were the first report of an altered APP secretion in fibroblasts from sporadic AD patients, extending to sporadic AD the concept that an altered APP metabolism may underlie the pathology and can be observed in peripheral cells as well as in the brain.

Fibroblasts represent also a model for the study of the effect of drugs potentially affecting APP synthesis and release. Moreover fibroblasts derived from AD patients allow the testing of drugs directly on a tissue expressing pathology-related defects in the metabolism of the precursor. Accordingly, we extended our experiments on the pharmacological modulation of sAPP release by testing the effect of bradykinin (BK) B2 receptor stimulation, which induced a dose-dependent increase of sAPP secretion (2.4 fold the basal) with an EC50 (2.8 nM) in the same order of magnitude as the Kd of BK for the receptor. At variance with PdBu the EC50 of BK in stimulating sAPP release was identical in AD and in controls. Notably, the effect of BK was independent from PKC, being insensitive to staurosporine inhibition. In contrast, fibroblasts from AD donors were greatly more sensitive to energy and oxidative metabolism inhibition. In fact the addition of sodium azide significantly inhibited sAPP secretion (maximal inhibition 51%, p<0.01) from AD patients at concentrations (10 mM) barely affecting sAPP release from controls.

Finally, the study of sAPP release from cells spontaneously overexpressing the protein, i.e. from patients with Down's syndrome (DS), showed that sAPP basal release was twofold that in age-matched controls while the pharmacological response to PdBu was blunted, indicating a saturation of this pathway due to the higher APP content and basal sAPP release. This observation suggests that the pharmacological modulation of sAPP secretion may differ in cells with altered APP expression, a notion relevant to the study of chronic pharmacological treatments potentially affecting APP synthesis.

The studied responses provide also important clues to the understanding of the mechanisms leading to altered sAPP secretion and processing in AD and disclose the possibility to affect its metabolism through pharmacological modulation in tissues derived from affected patients.

1.7 The amyloid-b protein of Alzheimer's disease increases acetylcholinesterase expression by increasing intracellular calcium in embryonal carcinoma P19 cells.

G. Sberna, J. Sáez-Valero, ÜK. Beyreuther, C.L. Masters, D.H. Small. Department of Pathology, The University of Melbourne, Parkville, Victoria, 3052, Australia. ÜCentre of Molecular Biology (ZMBH), University of Heidelberg, D-6900 Heidelberg, Germany. E-mail: javier@myriad.its.unimelb.EDU.AU

Keywords: acetylcholinesterase, amyloid, neurotoxicity, embryonal carcinoma, null

Abstract: One of the characteristic changes that occurs in Alzheimer's disease is the loss of acetylcholinesterase (AChE) from both cholinergic and non-cholinergic neurons of the brain. However, AChE activity is increased around amyloid plaques. The aim of this study was to examine the effect of Ab, the major component of amyloid plaques, on AChE expression. Ab peptides spanning residues 1-40 or 25-35 increased AChE activity in P19 EC cells. A peptide containing a scrambled Ab25-35 sequence did not stimulate AChE expression. To examine the possibility that the increase in AChE expression was mediated by an influx of calcium through voltage-dependent calcium channels (VDCCs), drugs acting on VDCCs were tested for their effects. Inhibitors of L-type VDCCs (diltiazem, nifedipine, verapamil), but not N- or P- or Q-type VDCCs, resulted in a decrease in AChE expression. Agonists of L-type VDCCs (maitotoxin, S(-) BAYK 8644) increased AChE expression. As L-type VDCCs are known to be modulated by cyclic AMP-dependent protein kinase, the effect of the adenylate cyclase activator forskolin was also examined. Forskolin stimulated AChE expression, an action that was blocked by the L-type VDCC antagonist nifedipine. The Ab25-35-induced increase in AChE expression was mediated by an L-type VDCC, as the effect was also blocked by nifedipine. The results suggest that the increase in AChE expression around amyloid plaques could be due to a disturbance in calcium homeostasis involving the opening of L-type VDCCs.

1.8 EXPRESSION AND ANALYSIS OF HEPARIN-BINDING REGIONS OF THE AMYLOID PRECURSOR PROTEIN OF ALZHEIMER'S DISEASE. S.S. Mok*, G. Sberna*, D. Heffernan*, R. Cappai*, D. Galatis*, H.J. Clarris*, W.H. Sawyerá, K. Beyreuther§, C.L. Masters* & D.H. Small*. Departments of *Pathology and áBiochemistry and Molecular Biology, The University of Melbourne, Parkville, Victoria, 3052, Australia and §Centre of Molecular Biology (ZMBH), University of Heidelberg, D-6900 Heidelberg, Germany. E-mail: s.mok@pathology.unimelb.edu.au

Keywords: heparin, proteoglycan, APP, amyloid, neurite

Abstract: The amyloid protein A-beta, is one of the major components present in amyloid plaques in the Alzheimer's disease brain. The 39-43 amino acid amyloid protein is cleaved from the larger amyloid precursor protein (APP) by a mechanism which is poorly understood. The physiological role of APP is also unclear. Several studies have demonstrated that APP plays a role in the regulation of cell growth and differentiation, and neurite outgrowth. APP has also been shown to bind heparan sulfate proteoglycans (HSPGs), suggesting that the interaction between APP and HSPGs may be important for its function. Previously, deletion mutagenesis studies by our group suggested that there are two domains within APP which bind heparan sulfate [1]. One domain is located in the cysteine-rich region between residues 18 and 190 [2]. The second domain was proposed to be rich in alpha-helix and lies between residues 311 and 493 (using the numbering system of APP695, ref. 3). To examine the heparin-binding properties of these two domains, the regions have been cloned and expressed in the methylotrophic yeast Pichia pastoris. Both recombinant proteins bound to heparin. The second domain was further characterized using peptides encompassing this region. Two peptides, homologous to APP316-346 and APP416-447were found to bind heparin. Circular dichroism studies indicated that APP416-447 shifted towards an alpha-helical conformation in the presence of heparin. This study demonstrates that APP contains two heparin-binding regions and it suggests that heparin-binding is associated with domains which have high secondary structure.

References
[1] Clarris H.J., Cappai R., Heffernan D., Beyreuther K., Masters C.L. and Small D.H. (1997) J. Neurochem. 68, 1164-1172
[2] Small D.H., Nurcombe V., Reed G., Clarris H.J., Moir R., Beyreuther K. and Masters C.L. (1994) J. Neurosci. 14, 2117-2127
[3] Kang J., Leaire H.G., Unterbeck A., Salbaum J.M., Masters C.L., Grzeschik K.H., Multhaup G., Beyreuther K. and Müller-Hill B. (1987) Nature 325, 733-736
Interaction of beta-amyloid peptide with aluminum in water solution: A molecular dynamics study. P. Carloni. Department of Chemistry, University of Florence, Via G. Capponi, 7, 50100 Florence, Italy. E-mail: paolo@risc1.lrm.fi.cnr.it

Keywords: aluminum, beta-amyloid peptide, molecular dynamics simulations, solution structure.

Abstract: A major component of the Alzheimer's disease's (AD)-specific brain lesions is the beta--amyloid peptide, a soluble, monomeric peptide composed of 39--43 aminoacids.

Aluminum, a risk factor in AD, enhances the aggregation and loss of secondary structure of the peptide, and subsequent nucleation and deposition processes (Fasman et al., Proc. Natl. Acad. Sci. USA 1995, 92, 369--371 ).

Using classical molecular dynamics simulations with the GROMOS code (van Gusteren et al., BIOMOS, Biomolecular Software, Laboratory of Physical Chemistry, ETH-Zurich), we have obtained a model for the solution structure of the aluminum--peptide adduct, that is not known experimentally.

Our calculations indicate that aluminum does not destabilize the secondary structure of the monomeric peptide, at least within the time of the simulation (1 ns). In particular, we have characterized a number of binding sites for the metal ion, which are both energetically and structurally similar. We propose that the formation of peptide--peptide cross links maybe the cause of the Al-promoted peptide aggregation found experimentally.