Previous research on brain tissue from 12 donors diagnosed as fAD [ 7 ] demonstrated significant accumulations of aluminum with 11 of the 12 brains having at least one tissue sample where the concentration of aluminum was defined as pathologically concerning (≥3.00 μg/g dry wt.). Aluminum-specific fluorescence microscopy [ 8 ] confirmed the presence of aluminum in these tissues and suggested that the majority of deposits of aluminum were extracellular associated with neuronal and cellular debris. A tentative suggestion was made that aluminum and amyloid-β were co-located in a senile plaque-like structure. Herein we have measured aluminum in brain tissue in Colombian donors carrying the fAD mutation PS1-E280A and compared the data with data for control brains. We have also used aluminum-specific fluorescence microscopy to investigate interrelationships between aluminum and amyloid-β in fAD.

An association between aluminum and amyloid-β in Alzheimer’s disease has been postulated for 40 years [ 1 ]. It has not been without controversy [ 2 ] though a consensus does now support the co-localization if not co-deposition of these two major risk factors in Alzheimer’s disease [ 3 ]. Familial Alzheimer’s disease, fAD, is characterized by genetic mutations affecting the expression and metabolism of the amyloid-β protein precursor (AβPP), leading to early onset of disease [ 4, 5 ]. One fAD mutation is PS1-E280A (Glu280Ala), a mutation that occurs in a significant cohort of individuals in Colombia. The mutation results in elevated cortical levels of amyloid-β, early disease onset (<50 years of age) and an aggressive disease etiology [ 6 ].

All chemicals were from Sigma Aldrich, UK unless otherwise stated. Paraffin-embedded brain tissue blocks mounted on Tissue Tek ® Uni-cassettes ® (Sakura Finetek Europe B.V., Netherlands), were cooled on wet ice for 10 min and sectioned at 5 μm via the use of a rotary RM2025 microtome using Surgipath DB80 LX low-profile stainless-steel microtome blades (both from Leica Microsystems, UK). Adjacent and numbered serial sections were floated out on ultrapure water (cond. <0.067 μS/cm) at 45°C and lifted onto SuperFrost ® Plus slides (Thermo Scientific, UK). Sections were subsequently dried at ambient temperature overnight. Dried sections were heated at 62°C for 20 min and allowed to cool to ambient temperature, immediately prior to use.

The aluminum content of tissues was measured by an established and fully validated method [ 9 ] that herein is described only briefly. Samples of cortex, between 0.6 and 2.0 g in weight, were thawed at room temperature and cut using a stainless-steel blade into sections approximately 0.3–0.5 g in weight. Tissues were dried for 48 h, to a constant weight, in an incubator at 37°C. Dry and thereafter weighed tissues were digested in a microwave (MARS Xpress CEM Microwave Technology Ltd.) in a mixture of 1 mL 15.8M HNO 3 (Fisher Analytical Grade) and 1 mL 30% w/v H 2 O 2 (BDH Aristar). The resulting digests were clear with no fatty residues and, upon cooling, were made up to 5 mL volume using ultrapure water (cond. <0.067 μS/cm). Total aluminum was measured in each sample by transversely heated graphite furnace atomic absorption spectrometry (TH GFAAS) using matrix-matched standards and an established analytical program alongside previously validated quality assurance data [ 9 ]. The latter included method blanks, detailed descriptions of which have been published recently [ 10 ].

Aluminum-specific fluorescence microscopy confirmed the presence of significant numbers of deposits of aluminum in brain tissue from all donors across all four lobes and in grey and white matter. Approximately two thirds of aluminum deposits were identified in grey matter. The majority of all deposits of tissue aluminum were shown to be extracellular and in most cases (71 out of 89), as confirmed using both ThS and Congo red, were co-located with amyloid-β. Tissue aluminum emitted characteristic orange fluorescence and was often senile plaque-like in size and appearance ( Figs. 1 A, 2A). Complementary staining of both same and serial sections using ThS and/or Congo red identified these aluminum-rich deposits as being additionally composed of amyloid-β ( Figs. 1 B, 2B) with merged images demonstrating intimate associations between amyloid and the metal ( Figs. 1 C, 2C). Congo red in combination with polarized light confirmed the presence of β-sheet amyloid-β in these plaque-like structures ( Figs. 1 D, 2D). In some structures, aluminum appeared to be associated with a core of amyloid-β ( Fig. 3 A–C) surrounded by a ‘nest’ of ThS-positive amyloid threads ( Fig. 3 C, D). There was also evidence of classical cerebral amyloid angiopathy where blood vessel walls stained positively for both aluminum and amyloid-β with the latter showing characteristic apple-green birefringence under polarized light ( Fig. 4 A–D). Occasional ThS-positive deposits were identified as spherulites by their classical Maltese cross signature under polarized light ( Fig. 4 C, D). Occasionally aluminum was identified in intracellular locations in tissues including in glia-like cells ( Fig. 5 A) and in neuronal axons ( Fig. 5 B).

The aluminum content of brain tissue across all donors and all brain regions ranged from 0.30 to 33.48 μg/g dry wt. ( Table 1 ). The majority of tissues (45 out of 83) had an aluminum content above 1.99 μg/g dry wt. with 14, 24, 10, and 35 tissues having contents in the range <1.00, 1.00–1.99, 2.00–2.99, and ≥3.00 μg/g dry wt. respectively. The median (IQR) aluminum content for each lobe was 2.04 (1.09–5.67), 2.19 (1.44–5.14), 2.11 (1.04–4.88), and 2.31 (1.02–6.48) for occipital, temporal, frontal, and parietal, respectively. There were no significant differences in aluminum content between lobes. There was no significant relationship between age of donor and aluminum content. There was a significant difference in aluminum content (median and IQR) between genders ( Supplementary Figure 1 ) with females (4.68; 3.22–6.95) having a higher content than males (1.62; 1.04–3.86) ( p = 0.01951).

DISCUSSION

The aluminum content of brain tissue from donors with the PS1-E280A fAD mutation was universally high with 42% of tissues having a concentration of aluminum above 3.00 μg/g dry wt., a content considered as pathologically significant [10]. Aluminum content in brain tissue in fAD was significantly higher than brain tissues from donors with neither clinical nor neuropathological diagnosis of neurodegenerative disease (Exley and Clarkson, unpublished). The new data confirm unequivocally the previous observation of very high brain aluminum content in fAD [7]. Aluminum-specific fluorescence microscopy identified aluminum in all brain tissues investigated (Figs. 1–5). The majority of all deposits of aluminum were extracellular and predominantly co-located with amyloid-β in primarily senile plaques and occasionally the vasculature. While the predominance of aluminum-amyloid deposits and the frequency with which aluminum and amyloid-β were observed together suggested their co-deposition, detailed scrutiny of the images favored their co-localization. For example, with either aluminum or amyloid-β acting as a nidus for the precipitation of the other. Deposits of both aluminum and amyloid were identified in the absence of each other. The former were found in a number of intracellular environments including in microglia-like cells and associated with neuronal axons. The presence of aluminum in axons in fAD brain tissues might imply an association with tau protein [14] though such remains to be confirmed.

This is the second study confirming significantly high brain aluminum content in fAD but it is the first to demonstrate an unequivocal association between the location of aluminum and amyloid-β in fAD. It shows that two prominent risk factors in the etiology of AD [15, 16] are intimately interwoven in the neuropathology of fAD. While there is a long history suggesting a role for aluminum in the provenance of senile plaques [17], the observation herein of the co-localization of aluminum and amyloid-β in vasculature has not previously been seen in either cerebral amyloid angiopathy [18] or autism spectrum disorder (Mold and Exley, unpublished). The association of aluminum and amyloid-β in brain tissue in neurodegenerative disease is not inevitable and so raises the question, what is different about fAD? The two distinct populations of fAD studied, previously [7] and herein, share a single characteristic in being prone to elevated levels of cortical amyloid-β early in life. This is attributed to genetic mutations associated with the metabolic machinery processing AβPP [1, 4, 5]. However, are the elevated levels of cortical amyloid-β, as opposed to cerebral spinal fluid amyloid-β [19], the direct consequence of enzymatic processing of AβPP or, perhaps, the indirect effect of elevated levels of brain aluminum? Might genetic predispositions underlying fAD be responsible for increased uptake and retention of aluminum in brain tissue with elevated amyloid-β being an indirect or even direct consequence of such. One could envisage increased amyloid-β in brain tissue as either an ameliorative response to high brain aluminum content or simply adventitious, aluminum acting as a nidus for the precipitation of amyloid-β. Either way it does seem indisputable that aluminum and amyloid-β are inextricably linked in the neuropathology of fAD.