Aluminum and Alzheimer’s Disease: An Update

Alzheimer’s disease (AD) is the most common form of dementia in the elderly. AD is characterized by senile plaques and neurofibrillary tangles (NFTs) comprised of amyloid-β protein (Aβ) deposits and hyper-phosphorylated tau protein (p-tau), respectively. Oxidative stress-induced neuronal damage is also involved in AD pathogenesis. In the 1970s, studies showed increased levels of aluminum (Al) in the AD brain, and neurofibrillary changes upon its injection into the brain, thus leading to the suggestion that Al may be one of the major causes of AD. However, later reports contradicted this hypothesis as studies revealed that Al-induced neurofibrillary changes were different from NFT sin AD, and intake of high dose Al-containing antacid drugs did not induce AD. Other in vitro and in vivo studies found that Al was neurotoxic, and possibly promoting aggregation of Aβ and p-tau. Here, we review and verify the validity of Al pathogenesis in AD. Despite the multitude of studies, no direct evidence currently exists that specifically links Al with AD pathogenesis. Therefore, more advanced cohort studies are necessary to better understand the absolute risk of Al for AD, and to rigorously compare this link using other neurotoxic metals. Taken together, Al may be an environmental factor promoting cognitive impairment in AD patients, as well as other free radical-generating metal ions such as iron, copper and zinc. By the 1970s, the association between Al and AD pathogenesis became a major topic of scientific discussion. Crapper and Dalton reported that administration of Al into cat brain induced memory impairment and neurofibrillary degeneration [15]. Increased levels of Al were documented in AD brain tissues [16-18]; however, these elevations were later found to be associated with aging and not with NFT levels [19]. Numerous studies have provided varying results (despite the use of similar methods) when measuring the levels of Al in human brain tissues, found to be both positive (Table 1) and negative (Table 2). Although the exact reason for these discrepancies is unclear, differences in the sample tissues may have been a factor. Indeed, since aging may increase Al levels in the brain [19], selection of age-matched control brain tissues may thus be important. Al was also suggested to be the cause of dialysis-associated encephalopathy (DAE) [20,21]. However, the DAE pathology was found to be clearly different from AD pathology (for more information, see below). Thus, in the following years, the debate about association between Al and AD diminished. Epidemiology of Al and AD Contamination of drinking water with Al polychloride in Camelford, England (Camelford water pollution incident; 1988) occurred. It was later revealed that the prevalence of dementia was not increased [22]. While, it was reported that the prevalence of AD was 1.5-fold higher in the area with water containing Al over 0.11 mg/L compared with the area of water containing Al under 0.01 mg/L [23]. Journal of Alzheimer’s Disease & Parkinsonism J o u r n a l o f A lzh eim ers ease & Prkin s o n i s m


Introduction
Alzheimer's disease (AD) is the most common form of dementia, mainly occurring in the aged population (>65 years of age). Pathologically, AD consists of two major hallmarks: neurofibrillary tangles (NFTs) and senile plaques (SPs), which are comprised of hyperphosphorylated tau (p-tau) filaments and amyloid-β protein (Aβ) fibrils, respectively. Numerous reports suggest that Aβ aggregation, namely the formation of Aβ oligomers, is an early event preceding other pathological changes in AD [1]. Aβ is physiologically generated from the cleavage of amyloid precursor protein by β-and γ-secretases in the endoplasmic reticulum (ER) and trans-Golgi network or in the late endosome, then secreted into the extracellular space [2]. Therefore, various factors promoting the formation of Aβ oligomers may be involved in the key molecular mechanisms underlying the pathogenesis of AD. For instance, in many forms of early-onset familial AD (FAD), highly aggregative Aβ42 is over-produced by mutant Presenilin 1/2genes [3]. Furthermore, Aβ aggregation may be promoted by apolipoprotein E [4] and by environmental factors, such as oxidative stress and diabetes [5]. Oxidative stress-inducing metal ions, such as iron (Fe) [6], copper (Cu) [7], zinc (Zn) and aluminum (Al) may contribute to the formation of SPs/NFTs and neuronal damage in the AD brain. Al has long been under debate as to whether it is a risk factor for AD. Therefore, the purpose of our review was to provide research findings on the putative link between Al and AD to understand what has been resolved thus far and what should be investigated in the future.

History of Al and AD
In the 1930s, Al was shown to have an adjuvant action [8], and in the 1940s, intrathecal injection of Al was reported to cause epilepsy in monkeys [9][10][11]. Klatzo et al. [12] showed that injection of a vaccine with Holt's adjuvant containing Al phosphate into the brains of rabbits caused epilepsy, as well as neurofibrillary changes. Terry and Pena [13] reported that NFTs observed in AD brains resembled those of Al-induced neurofibrillary changes; however, they later reported a morphological difference between the two [14]. Nevertheless, these earliest reports suggested the involvement of Al in AD pathology.
By the 1970s, the association between Al and AD pathogenesis became a major topic of scientific discussion. Crapper and Dalton reported that administration of Al into cat brain induced memory impairment and neurofibrillary degeneration [15]. Increased levels of Al were documented in AD brain tissues [16][17][18]; however, these elevations were later found to be associated with aging and not with NFT levels [19]. Numerous studies have provided varying results (despite the use of similar methods) when measuring the levels of Al in human brain tissues, found to be both positive (Table 1) and negative ( Table 2). Although the exact reason for these discrepancies is unclear, differences in the sample tissues may have been a factor. Indeed, since aging may increase Al levels in the brain [19], selection of age-matched control brain tissues may thus be important.
Al was also suggested to be the cause of dialysis-associated encephalopathy (DAE) [20,21]. However, the DAE pathology was found to be clearly different from AD pathology (for more information, see below). Thus, in the following years, the debate about association between Al and AD diminished.
Epidemiological research about the link between Al and dementia/AD continued in many other countries as follows.
A considerable number of epidemiological studies have been performed in industries associated with the manufacture of Al products, or in areas containing drinking water where Al was found. However, determining the occupational risk for AD has been difficult because of the limited size of sample numbers and difficulty of diagnosis [24]. Studies in the United Kingdom, Norway and Canada reported significant correlation between Al and AD [23-26] ( Table 3). Neri and Hewitt [27] reported that Al concentration over 0.2 mg/L in water may increase the incident rate of AD (odds ratio =1.46) in Canada [27]. However, in another study from Canada, fluorine content, rather than Al, was found to be more correlated with the incident rate of AD [28], and the Canadian Study of Health and Aging (CSHA) suggested a low risk for AD from the consumption of water containing Al [29]. Such discrepancies in the same country may have resulted from differing regions in which the studies were conducted. While, Jacqmin-Gadda et al. [30] reported that Al content may be associated with cognitive dysfunction in the regions with low silicon content and low pH in water. More recently, a cohort study in France (Personnes agees Quid, PAQUID) reported that Al concentrations over 0.1 mg/L in water increased the risk for AD (relative risk=3.04), and high silica concentrations in water decreased the risk for AD [31]. However, obtaining highly reliable data from such epidemiological studies is considered to be quite difficult because although the human diet contains Al, absorption and levels of Al in blood are altered by various factors, such as silicon, pH, fluoride, Fe, calcium (Ca), carboxylic acids and renal function [32,33]. Some reports have suggested no significant correlation between Al and AD ( Table 4). Bakar et al. [34] found no significant differences in the incidence of AD in areas with an acidic environment. Furthermore, many previous studies have revealed that consumption of tea or antacid drugs, which have high Al content, did not show a high incidence of AD [35][36][37][38][39][40][41][42]. These results indicate that various materials in drinking water and foods may act as the promoting or protecting factors for AD, and the effects of Al contained in water and foods may thus be masked by many factors.

Neurotoxicity of Al and Recommendation
Over the last 50 years, Al has been shown to be involved in epileptogenesis [9][10][11], and numerous in vitro and in vivo studies have reported on the neurotoxicity of Al. Al binds various organelles and proteins in the nucleus, cytoplasm, axon and synapse in neurons, exerting effects on the biological activity of magnesium, Fe, and Ca, thus interfering with the phosphorylation/dephosphorylation of nucleotides [43][44][45] [59][60][61][62]. However, the major pathological hallmark of Al neurotoxicity was found to be the hyper-phosphorylation of neurofilament proteins [63][64][65][66]. Indeed, this characteristic was found in brain tissue of DAE, which has implicated Al as its cause [67,68]. Owing to the high binding affinity of Al with phosphate, Al may thus promote aggregation of EAAS: electrothermal atomic absorption spectrometry; EDX: energy-dispersive X-ray spectroscopy; NAA: neutron activation analysis; ICP-MS: inductively coupled plasma source mass spectrometry; LMMS: laser microprobe mass spectrometry; ICP-AES: inductively coupled plasma atomic emission spectrometry; INAA: instrumental neutron activation analysis; IF-CLM: immunofluorescence using confocal laser microscopy; HS: histological staining. EAAS: electrothermal atomic absorption spectrometry; EDX: energy-dispersive X-ray spectroscopy; STIM: scanning transmission ion microscopy; ICP-MS: inductively coupled plasma source mass spectrometry.  hyper-phosphorylated neurofilaments. However, evidence of such an effect may not directly indicate that Al plays a pivotal role in AD pathogenesis because NFTs in AD consists of p-tau.
Owing to the lack of consensus amongst epidemiological studies, it is still unclear whether oral Al intake increases the risk for AD. Therefore, this risk has not been approved by the European Food Safety Authority [32], Joint Food and Agriculture Organization of United Nations (FAO)/World Health Organization (WHO) [33], and Bundes institut für Risikobewertung. However, because aging is the major risk factor for sporadic AD, and that aging is a strong factor for promoting Al accumulation in brain tissue [19, 69,70], the likelihood that Al is a risk factor for sporadic AD may thus be increased in the elderly. Impairment of the blood brain barrier or renal dysfunction during aging may promote the accumulation of Al in the brain. However, to study such effects may be quite difficult because aging itself is the strongest risk factor for AD, and some radical-generating metals other than Al, such as Fe, Cu and Zn may also facilitate Aβ oligomer formation and accelerate AD pathogenesis [71,72]. Although it is difficult to determine the safe doses of Al intake, the FAO/WHO presently recommend a tolerable weekly intake of 2 mg /kg body weight [33].

Does Al Associate with AD Pathology?
Whether Al is involved in the onset and progression AD remains an important issue. Many previous reports have suggested that various molecular mechanisms contribute to AD pathogenesis. To date, the widely accepted major factors are: Aβ (particularly Aβ oligomers) aggregation, phosphorylation/aggregation of tau protein (forming NFTs), oxidative stress, mitochondrial damage and synaptic dysfunction [73]. Therefore, the influence of Al on these pathological characteristics of AD has been explored. Studies have indicated that Almay be associated with the aggregation of Aβ [74,75] and p-tau [74][75][76][77][78][79][80], and may also be involved in Aβ deposition [81][82][83][84]. Al accumulation was shown to not consistently coincide with NFTs (Table 5) and SPs (Table  6). Furthermore, the concentration of Al in biological fluids is less than 1 µM (Bush AI, personal communication). Al accumulation was shown to not consistently coincide with NFTs (Table 5) and SPs (Table 6). Thus the role of Al in the formation of NFTs and SPs compared with other metal ions, such as Zn, Cu and Fe may not be as prominent. Zn is a key modulator for synaptic neural transmission, reaching 150-300 µM during synaptic activity, thus possibly contributing to Aβ aggregation [85]. Also, trace amounts of Cu were shown to induce Aβ accumulation in brain tissues of cholesterol-fed rabbits [86]. Therefore, the definitive involvement of Al in NFT/SP formation in the AD brain is difficult to elucidate. Nevertheless, Al itself may promote neuronal damage independently of NFT/SP.
As previously mentioned in this review, the Al hypothesis for AD was based on a pathological similarity of neurofibrillary degeneration seen in both the AD and DAE brain. However, Reusche et al. [87] found that in brain tissue from dialysis patients, only 1 case out of 50 exhibited the accumulation of Aβ42 and p-tau. Phosphorylated neurofilaments and cytoplasmic argyrophilic inclusions have been shown to be characteristics of Al-induced pathology in DAE rather than the paired helical filaments that are consistently observed in AD [67,68,87]. Remarkably, Al-containing lysosome-derived argyrophilic inclusion in neurons and glial cells is a characteristic of DAE in those brain tissues. Overall, pathological findings between AD and DAE are different.  EDX: energy-dispersive X-ray spectroscopy; WDX: wavelength-dispersive X-ray microanalysis; SIMS: secondary ion mass spectrometry; TEM-EDX: energydispersive X-ray spectroscopy combined with transmission electron microscopy.

Does Al Promote Dementia in AD Models?
Numerous studies of transgenic (Tg) animal models of AD have been performed to further confirm the possibility that Al is not directly linked to AD, but may enhance cognitive dysfunction and/or AD pathology. Pratico et al. [88] fed Tg2576 mice an Al-enriched diet (2 mg/kg/day, from 3-12 months old), finding that levels of isoprostane and Aβ increased and Aβ deposition was enhanced (compared with wild-type [wt]). Furthermore, vitamin E, an anti-oxidant, reversed such responses, which may support the idea that Al accelerates oxidative stress and AD pathology. Ribes et al. [89] fed Tg2576 or wt mice with a low dose of Al lactate (1 mg/g, from 5 months for 120 days), and found impaired cognition via oxidative stress in wt, which may not have interacted with the amyloidogenic pathway. Tg2576 mice fed the same diet (5-11 months old) showed that Al may act as a pro-oxidant agent in both Tg2576 and wild-type mice [90]. Al levels have also been shown to be higher in the hippocampus than in the cerebellum and cortex in both Tg2576 and wt mice [91]. Altogether, these studies have revealed that long-term exposure on a diet containing Al may induce oxidative stress and cognitive impairment with or without an exacerbated AD pathology. Recently, Akiyama et al. [92] found that administering Al or Zn in drinking water (100 mg/kg/day, from 8 months for 4-10 months) to Tg2576 or Tg2576/tau-P301L mice did not enhance the deposition of Aβ or tau deposition, nor the increase insoluble Aβ or Aβ oligomers. Although cognitive function and oxidative stress markers were not analyzed in the study, it is evident that an intake of such a high dose of Al did not enhance AD pathology related to the mutation of amyloid precursor protein. Overall, it is unclear as to why these studies have shown contradictory results. Our re-evaluation of these previous studies of animal experiments based on the differing types of Al salts used did not account for the discrepancies.
Furthermore, the Tg2576 model is an early-onset FAD model. Therefore, the effects of Al intake on AD pathology and cognitive function remain to be observed in a model of the late-onset sporadic AD. Unfortunately, such animal models are currently not available. Interestingly, however, chronic intake of low dose Al in aged wt rats was shown to cause cognitive deterioration and neuronal loss in regions usually affected by AD, the entorhinal cortex and hippocampus [93]. Therefore, Al intake may not be a strong factor for the cause or promotion of early-onset FAD pathogenesis. Nevertheless, long-term intake of low dose Al may promote oxidative stress and neuronal damage, thus contributing to the cognitive decline in the elderly population irrespective of their risk for developing this disease. Such a possible effect may not be specific for Al, and other radical-generating metals may also contribute to the cognitive decline.

How Can We Understand the Real Role of Al?
There are a number of environmental factors (e.g. aging, ischemia, diabetes, trauma, smoking) that pose risks for AD and cognitive decline [94]. As reviewed here, Al may not be the major environmental risk factor for the onset and progression of AD. However, Al might contribute to the acceleration of cognitive decline, as well as other free radicalgenerating metals, in the elderly population irrespective of whether they have AD. To better understand this hypothesis, a large cohort study, rather than an epidemiological investigation, may be necessary, for example the worldwide AD Neuroimaging Initiative (ADNI) [95]. Because brain Aβ accumulation may start 10-20 years before the onset of mild cognitive impairment or the prodromal AD stages [96], it may thus be necessary to evaluate the involvement of free radical-generating metal ions (Fe, Cu, Zn and Al) during these stages. Furthermore, levels of these metals in the cerebrospinal fluid (CSF) should be measured, followed by correlation analysis with amyloid imaging, AD biomarkers in the CSF, and cognitive examination outcomes.

Conclusion
Previous epidemiological, biochemical, and pathological studies have provided quite conflicting results regarding the involvement of Al in AD pathogenesis. Findings from studies involving DAE and AD mouse models therefore suggest that Al intake is not a direct cause for AD. However, Al and other free-radical generating metals show neurotoxic activity in vitro, thus indicating that they may promote cognitive impairment in rodents and humans because these metals accumulate in the aging brain. Therefore, above normal intake of Al (and other metals) is an environmental factor that may promote cognitive decline in the aged population, irrespective of whether they are at risk for developing AD. More advanced cohort studies are thus necessary to understand the absolute risk of Al (and other metals) for AD.