Walton 2013

Journal of Alzheimer’s Disease 35 (2013) 7–43 7
DOI 10.3233/JAD-121909
IOS Press
Review
Aluminum’s Involvement in the Progression

of Alzheimer’s Disease
J.R. Walton∗
Faculty of Medicine, University of New South Wales, St George Hospital Campus, Sydney NSW, Australia
Accepted 10 January 2013
Abstract. The neuroanatomic specificity with which Alzheimer’s disease (AD) progresses could provide clues to AD etiopathol-
ogy. Magnetic resonance imaging studies of AD clinical progression have confirmed general conclusions from earlier studies
of AD neuropathological progression wherein neurofibrillary tangle pathology was observed to spread along a well-defined
sequence of corticocortical and corticosubcortical connections, preferentially affecting certain cell types, while sparing others.
Identical and non-identical twin studies have consistently shown AD has mixed (environmental and genetic) etiopathogenesis.
The decades-long prodromal phase over which AD develops suggests slow but progressive accumulation of a toxic or infective
agent over time. Major environmental candidates are reviewed to assess which best fits the profile of an agent that slowly accrues
in susceptible cell types of AD-vulnerable brain regions to toxic levels by old age, giving rise to AD neuropathology without
rapid neuronal lysis. Chronic aluminum neurotoxicity best matches this profile. Many humans routinely ingest aluminum salts
as additives contained in processed foods and alum-treated drinking water. The physical properties of aluminum and ferric iron
ions are similar, allowing aluminum to use mechanisms evolved for iron to enter vulnerable neurons involved in AD progression,
accumulate in those neurons, and cause neurofibrillary damage. The genetic component of AD etiopathogenesis apparently
involves a susceptibility gene, yet to be identified, that increases aluminum absorption because AD and Down syndrome patients
have higher than normal plasma, and brain, aluminum levels. This review describes evidence for aluminum involvement in AD
neuropathology and the clinical progression of sporadic AD.
Keywords: Aluminum compounds, Alzheimer’s disease, amyloid-␤ protein precursor, animal, causality, disease models,
intestinal absorption, neurofibrillary tangles, neurotoxicity syndromes, translational medical research
INTRODUCTION Epidemiological studies for AD, based on identical

and non-identical twin pairs, have consistently shown
One of the most important and challenging problems that AD causality has significant environmental and
in contemporary clinical neuroscience is to explain the genetic components. If one member of an identical twin
neuroanatomic specificity of neurodegeneration that pair develops AD while the other remains AD-free, the
occurs in Alzheimer’s disease (AD) [1]. That is, to discordance is attributable to an environmental compo-
understand why certain large neurons, particularly in nent of AD causality. If identical twins show a higher
the superficial entorhinal cortex, are among the ear- rate of concordance for AD than non-identical twins,
liest and most severely-damaged cell populations to this provides evidence for a genetic component of AD
be affected in AD. An understanding of this selective causality.
damage may provide important clues for unraveling A nationwide Finnish twin cohort study [3] showed
AD causality [2]. more than two-thirds (68.7%) of identical twin pairs
were discordant for AD, indicating a significant envi-
∗ Correspondence to: Dr. J.R. Walton, E-mail: j.walton@unsw. ronmental component for AD causality. The same
edu.au. study showed a probandwise concordance rate for AD
ISSN 1387-2877/13/$27.50 © 2013 – IOS Press and the authors. All rights reserved
This article is published online with Open Access and distributed under the terms of the Creative Commons Attribution Non-Commercial
License.
8 J.R. Walton / Aluminum’s and Alzheimer’s Disease Progression
of 31.3% in identical twin pairs compared to 9.3% prodromal phase of AD, comparing brain scans of
in non-identical twin pairs, also indicating genetic apparently normal individuals with a family history
involvement. Twin-based studies have consistently of AD, which confers an increased risk for this con-
shown that AD has mixed causality with significant dition, and apparently normal individuals without a
environmental and genetic components [3–5]. family history for AD [7]. The second study takes place
Many AD researchers have focused on the genetic near the end of the prodromal period and compares
component of AD which is currently believed to MRI scans of subjects with (amnestic) mild cognitive
involve aberrant metabolism of the amyloid-␤ pro- impairment (aMCI) before and after they convert to
tein precursor (A␤PP) and its amyloid-␤ (A␤) peptide overt AD, with scans performed over the same time
cleavage product. Attention has more recently been period from healthy controls [8]. The third study com-
directed toward the environmental component of AD. pares two brain scans, separated by at least one year,
Conventional viruses, prions, and metal neurotoxi- from groups of patients diagnosed with probable AD,
cants, in particular soluble forms of lead, mercury, and and age-matched non-demented controls, to demon-
aluminum, have all been proposed for this role. We strate the gray matter atrophy that occurs over time in
will consider how well these environmental candidates specific regions of AD-affected brains [9].
fit the profile as the environmental component of AD
etiopathogenesis and attempt to understand how that Family history and increased risk of AD
component relates to AD progression.
The present review article has five parts, describ- An MRI/VBM study aimed to measure atrophic
ing: (1) the anatomical sequencing of interconnected change in gray matter of cognitively-normal individ-
brain regions involved in clinical and neuropatho- uals with increased risk for AD due to family history
logical AD progression; (2) cell and tissue features (FH+) [7]. The study included 11 subjects with a mater-
proposed to enhance the propensity of certain cell nal history of AD (FHm), 10 with a paternal history of
types in AD-vulnerable brain regions for neurofibril- AD (FHp), and 32 without any parental history of AD
lary tangle (NFT) formation; (3) a comparison of the (FH-). Offspring with a family history of AD, particu-
major candidates for the environmental component of larly from the maternal side, have been shown to have a
AD; (4) evidence that the environmental component 4- to 10-fold increased risk for late-onset AD compared
participates in AD neuropathology; and (5) how the to those without a family history of AD [10].
environmental component may interact with cell and The prospective subjects, recruited from a referral-
tissue features to increase neuronal propensity for NFT based memory clinic, were given a standard diagnostic
formation and its role in AD progression. evaluation. A neurologist used the Clinical Dementia
Rating (CDR) scale and the Mini-Mental State Exam-
ination (MMSE) to assess the subjects for dementia.
LONGITUDINAL STUDIES OF AD Subjects were aged 63–83 years at baseline and had at
CLINICAL PROGRESSION least 12 years of education. Controls without a family
history for AD had a CDR score of zero and a MMSE
Magnetic resonance imaging (MRI) allows spe- score of 28 or higher. The subjects were given medical,
cific brain regions to be mapped in living patients. neuropsychological, and MRI assessments at baseline
Brain mapping software, in particular voxel (3D pixel) and at the 2-year follow-up. Gray matter, white matter,
based morphometry (VBM) [6], can effectively com- and cerebrospinal fluid (CSF) volumes were measured
bine multiple MRI scans from a single individual, on both occasions and VBM was used to superimpose
taken on several occasions over a period of time, to the MRI scans in order to assess them for change.
detect rates of atrophy in particular regions of the indi- Baseline MRI data already revealed significantly
vidual’s brain. Alternatively, MRI/VBM can combine greater loss of gray matter and CSF expansion in
brain scans from a population of patients at particular FH+ subjects than in FH− subjects [7]. The FH+ sub-
stages of a disease to determine average gray matter jects had significantly more gray matter atrophy than
volumes at those stages. For AD, cognitive testing of FH− subjects in the parahippocampus (including the
the same subjects is generally carried out close in time entorhinal cortex), hippocampus, anterior cingulate,
to their MRI scans. medial frontal cortex, and precuneus (p < 0.001). When
Three examples are described here of longitudi- the family history group was split into FHm and FHp
nal MRI/VBM studies carried out at different stages sub-groups, only the maternal group showed an asso-
of AD. The first of these studies takes place in the ciation with increased whole brain atrophy. Those in
J.R. Walton / Aluminum’s and Alzheimer’s Disease Progression 9
the FHm group with an APOE4 allele exhibited signif- the entire hippocampus. Parietal lobes also showed
icantly more atrophy in the frontal cortex [7]. atrophy whereas the frontal lobes were relatively
At the 2-year follow-up visit, further atrophy was spared [8]. These results show that significant gray
observed in the left precuneus and the left parahip- matter atrophy occurs before AD is diagnosable.
pocampal gyrus. Atrophy had also increased in the The 33 aMCI subjects were ultimately diagnosed
right hippocampus, right precuneus, bilateral middle with AD at the time of their third scan. Gray matter
temporal gyrus, anterior cingulate, and posterior cin- atrophy in the converted AD group’s brains was more
gulate of those with a positive family history for AD extensive throughout the medial temporal lobes and
compared to those without the family history [7]. the temporoparietal association neocortices than in the
scan taken one year earlier. By this time, gray matter of
the frontal lobes showed substantial atrophy for the first
Conversion from aMCI to AD time, particularly in anterior frontal lobes and superior
frontal gyri. Some gray matter loss was also evident in
The second MRI/VBM study analyzed gray matter the midbrain [8]. Other authors confirm that subjects
loss that occurs in specific brain regions during conver- who convert from aMCI to AD have significantly less
sion from aMCI to AD [8]. The study was preceded by hippocampal and amygdala gray matter volume and
data collection from 61 subjects with aMCI who pre- less temporal and parietal lobe cortical thickness than
sented for MRI scans on three occasions over a period non-converters [11].
of time lasting approximately three years. Criteria Cognitive test scores for the aMCI/AD group also
for aMCI were: memory complaint, impaired mem- changed over the period during which the three scans
ory for their age, almost normal cognitive function, were obtained: MMSE scores fell from 27 to 25 to 24,
fairly normal activities of daily living, and absence of decreasing 0.9 points per year; CDR scores rose from
dementia. 1.0 to 2.0 and 3.5, increasing 0.7 points per year; and
An inclusion criterion for the MRI/VBM analysis Dementia Rating Scale scores decreased from 132 to
was that the patients with aMCI converted to AD 125 to 120 or 3 points per year over the span of this
around the three-year timeframe. Hence, the study period [9]. Other investigators have reported that atro-
included 33 subjects with aMCI, who all converted to phy of the anterior hippocampus coincides in subjects
AD at a similar rate and had three MRI scans performed with a CDR of 0.5 whereas atrophy of the posterior
during the course of conversion, which occurred over hippocampus is only seen in subjects with a CDR of 2
a median interval of three years. The 33 aMCI subjects or 3 [12].
were compared with 33 healthy controls [8].
The first of three scans was performed approxi-
mately 3 years prior to the diagnosis of AD. The second Maps of gray matter atrophy in AD brains
was about one year before AD diagnosis and the third
scan was done at the time of AD diagnosis. At each The third example involves MRI/VBM mapping of
time point, all 33 scans for each group were combined the spreading loss of gray matter, imaged on two occa-
with VBM software and templates to determine aver- sions separated by approximately 1.5 years, averaged
age gray matter volumes for the aMCI/AD group and from 12 patients diagnosed with probable AD com-
the control group. pared to 14 healthy elderly controls [9]. Novel brain
At the time of the first scan, the averaged gray mapping methods combined the corresponding corti-
matter loss in the aMCI cohort was primarily con- cal regions of all subjects to create average maps that
fined to the anterior medial and inferior temporal lobes could be visualized in high resolution.
(bilateral entorhinal cortex, anterior hippocampus, and The resulting maps differentiated AD from normal
left amygdala) with some involvement of the fusiform aging (controls) and were able to distinguish differ-
gyrus. The posterior hippocampus was relatively unaf- ent phases of AD. The temporal sequence of deficits
fected. Gray matter loss was bilateral, affecting the left that occurs as AD spreads across the cortex was visu-
hemisphere slightly more than the right hemisphere alized and related to cognitive decline with MMSE
[8]. scores. The serial MRI images of AD brains indicated
At the time of the second scan, the averaged gray gray matter loss had spread from limbic and tempo-
matter atrophy in the aMCI group was greater in the ral cortices into frontal and occipital brain regions
medial temporal lobes, inferior temporal lobes, and while sparing sensorimotor cortices. Some local rates
posterior regions of the temporal lobes, now including of gray matter loss were more than 5% per year in
AD as opposed to 0.9% per year in controls. The left Braak staging of AD neuropathological
hemisphere was affected significantly earlier than the progression
right hemisphere. Frontal regions were spared early
in the disease but eventually showed more than 15% In 1991, Braak and Braak published a study in
loss [9]. which they examined 83 human brains with varying
degrees of AD involvement, including 21 from elderly
demented humans whose brain tissue had sufficient
Relationship between AD clinical progression and densities of NFTs to confirm their clinical diagnosis
neuropathological progression of AD [13]. The study concluded that NFT pathology
follows a predictable pattern that allows AD-affected
Results from MRI/VBM studies provide quantita-
brains to be categorized into six stages: Stages I–II
tive, dynamic visualization of cortical atrophy in living
or transentorhinal cortex stages are pre-clinical. (The
subjects with aMCI and AD and have the advantage
transentorhinal cortex is in the perirhinal cortex where
that they can be supplemented by cognitive testing. The
it interfaces with the entorhinal cortex.) [1]; Stages
MRI/VBM studies confirm that gray matter atrophy
III–IV or limbic stages are characterized clinically
occurs in a well-defined sequence with AD progression
by incipient AD; and Stages V–VI or neocortical
in a manner that is fundamentally consistent with the
stages are characterized by overt AD (Table 1). Studies
temporal sequence previously predicted by studies that
of severe AD are complicated by wide-scale neuron
relied on postmortem specimens of neuropathologi-
loss and increasing neuropathology in glia and non-
cal deterioration in AD-affected brain regions. Both
pyramidal cells [22]. The Braak hierarchical model
approaches have been valuable for analyzing AD pro-
of AD neuropathology has been critically tested and
gression from one region to another in the aging brain.
validated by Gertz et al. [23].
SEQUENCING THE BRAIN REGIONS Cell–cell connectivity and AD neuropathological

INVOLVED IN AD NEUROPATHOLOGICAL progression
PROGRESSION
Pearson et al. showed that cell–cell connectivity
Several neuropathological studies, briefly presented plays an essential role in AD progression [14]. More
here, have identified brain regions specifically involved specifically, they observed that AD neurodegenera-
in AD and attempted to determine either the order in tion commences in olfactory structures located in the
which they are affected in AD progression or the extent medial temporal lobe and then spreads widely via
of severity to which they are affected [2, 13–17]. Two anatomical connections to more distant cortical regions
others examined how many A␤ deposits and NFTs (Table 1).
form in brains of older non-demented subjects, rela- Deafferentation of corticocortical fibers in one brain
tive to their ages [18, 19]. Another identified brainstem region may result in NFT damage within the region
regions also involved in AD [20]. to which those corticocortical fibers normally project
These studies originally analyzed the numbers and [14]. NFTs appear to develop in conjunction with
distributions of both NFTs and neuritic plaques (NPs) denervation of short feed-forward and/or feed-back
in order to determine which AD hallmark is more suited corticocortical fibers as well as the ascending or
as a biomarker of brain regions involved in AD pro- descending paths. The number of pyramidal cells with
gression. All studies ultimately used NFT numbers for NFTs in layer V of the middle temporal neocortex is
evaluating the timing and/or severity of AD-affected in a 2 : 1 ratio with NFT-containing pyramidal cells in
brain regions. Reasons for this choice are that NFTs layer III. Moreover, the NFTs of layers III and V are
develop within cells located in specific laminae within arranged in clusters that are in register with each other.
well-defined brain regions [13]. The characteristic dis- There is close correspondence between the distribution
tribution pattern of NFTs permits the differentiation of NFTs in the neocortex and damage in anatomically
of stages in AD progression. In contrast, NPs have connected regions [14].
a patchy distribution pattern within discrete architec-
tonic units, significant variation from one brain to NFT counts indicate AD-affected brain regions
another, and multimodal formation [13]. Also, unlike
NFTs, A␤ correlates poorly with rates of brain atrophy The cells of origin for the perforant pathway, located
[21]. in layers II and III of the superficial entorhinal cortex,
Table 1
Sequences of NFT-affected brain regions, with regard to timing or severity, in neuropathological AD progression
Braak stages Braak and Braak, 1991 [13] Arnold et al., 1990 [15] Kovács et al., 2001 [17] Pearson, 1996 [14]
Stage I transentorhinal cortex entorhinal cortex olfactory bulb piriform cortex
Stage II entorhinal cortex, CA1 subiculum/CA1 complex anterior olfactory entorhinal cortex
nucleus
Stage III amygdala, basolateral nucleus temporal pole cortex entorhinal cortex amygdala
anterodorsal thalamus perirhinal cortex periamygdaloid cortex agranular insula
reunions nucleus, hypothalamic amygdala, access basal nucleus CA1 field orbitofrontal area
tuberomamillary nucleus post parahippocampal cortex anterior amygdala CA1/subicular complex
basal magnocellular complex nucleus basalis of Meynert gyrus rectus perirhinal cortex
Stage IV accumbens nucleus, putamen anterior insula lateral orbitofrontal cx assoc areas anterior
temporal gyri
corticomedial amygdala mid and inferior temporal cortex occipital cortex assoc areas cingulate cortex
basal claustrum, striatum non-primary association cortex cingulate cortex temporal pole cortex
Stage V parasubiculum, transsubiculum superior temporal cortex assoc area inferior parietal
subiculum, CA4, putamen orbitofrontal cx inferior parietal
all neocortex association areas fusiform cortex dorsolateral prefrontal assoc
areas
anterobasal insula, uncus primary sensory assoc cortex post assoc areas temp lobe
orbitofrontal cortex retrosplenial cortex post assoc areas sup parietal
lobe
substantia nigra pars complex ventral ant & post cingulate frontal pole
cortices
thalamus, anteroventral nucleus dorsal cingulate cortex posterior prefrontal cortex
orbitofrontal cortex dorsal prefrontal cortex
hypothalamic lateral tuberal agranular cortex
nucleus
Stage VI fascia dentata primary sensory areas
extrapyramidal system
collect incoming information from sensory associa- AD neocortex [26]. The laminar distribution of NFTs
tion cortices, which is relayed to the dentate gyrus and in all neocortical association areas favors layers III and
CA1/subiculum zone of the hippocampal formation. V with relatively few NFTs appearing in cortical layers
These cells contain more NFTs than cells in any other II, IV, and VI [14, 15].
of the 30 cortical regions of the AD brain examined NFTs and NPs have different distribution patterns,
in a study by Arnold et al. [15] (Table 1). They are with NFTs being more selectively distributed than NPs
selectively vulnerable during aging and MCI as well [15]. Most NPs are found in neocortical layers II, III,
as AD, suggesting they become damaged at a very early IV, and V [14]. The selective involvement of NFT
stage [24]. Other large pyramidal cells, in layer IV of damage in neurons of layers III and V of the asso-
the entorhinal cortex, are also heavily invested with ciation cortices is expected to disrupt corticocortical
NFTs, but at a later stage than those in the superficial and corticosubcortical connections [14, 15, 27].
entorhinal cortex. By endstage AD, all cell layers of
the entorhinal cortex are devastated [1]. Olfactory deterioration in AD neuropathological
The hippocampal CA1/subiculum zone is also progression
severely affected by NFT formation whereas other
areas of the hippocampal formation are largely spared. Kovács and colleagues [16, 17] counted NFTs in
The entorhinal cortex/hippocampus/amygdala regions olfactory structures to link the olfactory bulb and
are the most severely affected in AD, on the basis of related olfactory regions to the Braak hierarchical
NFT numbers, whereas the motor and primary sensory model for AD staging [16], and to explain the well-
cortices are the least affected [15]. Many more NFTs known observation that olfaction is impaired at an early
are located in limbic and temporal lobes than in the stage of AD (Table 1). They concluded that: (1) the
parietal, frontal, and occipital lobes. Neurons in lay- olfactory bulb is one of the first structures to develop
ers II and IV of the entorhinal cortex have substantial NFTs; (2) this may occur prior to NFT damage in the
afferent and efferent interconnections with temporal entorhinal cortex; (3) NFTs are observed in olfactory
and posterior parietal higher order neocortical associ- bulbs at Braak stages 0 or I; and (4) impaired olfaction
ation areas [25], the most NFT-damaged regions in the in early stages of AD is attributable to early NFT dam-
age in the olfactory bulb and anterior olfactory nucleus The view expressed by Kovács et al. [16] that the
[16, 17]. olfactory region is affected by AD neuropathology
prior to the entorhinal cortex is countered by the argu-
ment that AD affects the entorhinal cortex before the
NFTs in subcortical nuclei olfactory mucosa or olfactory bulb [18]. According
to Ulrich, NFTs are sometimes seen in the entorhi-
Pyramidal neurons of neocortical layer V project to
nal cortex and/or hippocampus while absent from the
the striatum, the nucleus basalis of Meynert, and the
olfactory bulb. However, it is never the case that NFTs
brainstem. The nucleus basalis is virtually devoid of
are present in the olfactory bulb while absent from the
tangles in brains from non-demented individuals under
entorhinal cortex and/or hippocampus [18].
age 75 years and shows only a few NFTs in brains of
The hippocampus, entorhinal cortex, and nucleus
older controls [19]. The nucleus basalis of Meynert is
basalis of Meynert contain fewer NPs per field than
highly variable between AD cases, some being barely
any other region [15]. The entorhinal cortex has one of
affected and others severely affected by NFTs [15].
the lowest NP densities, and most NPs in this region are
Certain brainstem nuclei are selectively vulnerable
located in layer III. By the time AD is evident, entorhi-
to AD [20] and their deterioration may give rise to
nal cortical cells of origin for the perforant pathway are
additional features of AD. The dorsal raphe complex
heavily invested with NFTs with many in the form of
is severely invested by NFTs in most AD cases. The
extracellular “ghosts” [25]. Damage to the perforant
dorsal raphe complex consists mainly of serotonergic
pathway involves deterioration of its glutamatergic
neurons and accounts for a substantial proportion of
terminals that normally innervate the hippocampal for-
the major ascending serotonergic projections to the
mation. This ultimately results in disconnection of the
forebrain. Dorsal raphe pathology could account for
hippocampal formation from the entorhinal cortex and,
the significant decrease in serotonergic innervation and
hence, the neocortex [29]. A layer of NPs forms in
serotonin decrease in the CSF of AD patients [20].
the center of the dentate gyrus molecular layer, pre-
The locus coeruleus is also severely affected in AD.
cisely where the terminals of the perforant pathway
Damage occurs in the locus coeruleus at a very early
were previously located [29, 30].
stage of AD, possibly preceding damage in the transen-
The amyloid cascade hypothesis assumes that A␤
torhinal cortex [28], a finding that should be examined
deposits precede NFTs and cause their formation [31].
with MRI/VBM. This nucleus has major noradrener-
A␤ deposition is much more prominent in the neocor-
gic projections to basal forebrain nuclei and several
tex of controls and AD cases than in limbic regions [14,
cortical regions. Pathology in the locus coeruleus may
16]. The average A␤ plaque density in the temporal,
underlie disorders of selective attention and neural con-
insular, and orbital cortices is three-fold greater than
trol of sleep and wakefulness [20].
in the hippocampal CA1 field. Also, A␤ plaques were
observed to have formed in the temporal neocortex
NFTs and Aβ deposits: Which occurs first in AD? prior to neocortical NFTs [19].
However, these findings are insufficient to support
A search for NFTs and A␤ in limbic regions from the concept that A␤ deposition precedes and causes
non-demented subjects under age 65 revealed that 22 NFTs. Studies of AD progression have shown that the
(58%) of the 38 brains showed AD change in the form limbic regions which, on average, exhibit AD neu-
of NFTs only [18]. Three (8%) seemed to have only A␤ ropathology 25 years earlier than neocortical regions
deposits and 13 (34%) had both NFTs and A␤ deposits. [32] show NFTs in most brains earlier than A␤ deposits
Almost all A␤ deposits observed in brains of these non- [16, 18]. The occurrence of A␤ plaques preceding
demented middle-aged humans were primitive. Classic NFTs in the neocortex raises the possibility that neo-
plaques with cores were seldom observed [18]. These cortical A␤ plaques could form in response to NFT
findings [18] are very similar to findings from a study damage from layer IV neurons of the entorhinal cortex
by Kovács et al. [16] who reported that all 15 AD cases and/or from AD-affected hippocampal CA1 neurons
and 13 (87%) of the 15 aged controls they examined that normally project to the neocortex, rather than from
exhibited NFTs in the olfactory bulb whereas only 5 neocortical sources. For example, the hippocampus has
(33%) had A␤ deposits and these were diffuse. Com- stronger feed-forward output to the medial temporal
pact and neuritic A␤ plaques were not seen [16]. Thus, gyrus than feedback from the medial temporal gyrus
NFTs appear earlier than A␤ deposits in olfactory and [33]. Thus, NFT-induced damage in the hippocampus
limbic regions, even in controls [16, 18]. could result in A␤ plaques in the medial temporal
gyrus. Tau pathology in limbic regions precedes A␤ A consensus on AD neuropathological progression

pathology by at least 27 years [22, 34]. Immunos-
tained tau is visible in brain tissue of many children The Braak hierarchical model for AD progression
and young adults despite the absence of evidence for and other studies described above support the con-
immunoreactive A␤ [22]. cept that, toward the end of a long prodromal phase,
NFT neuropathology is already spreading in a stepwise
manner along well-defined corticocortical connections
Connectivity and disconnectivity in AD between the entorhinal cortex and other limbic struc-
tures. More brain regions become involved as AD
AD-affected brain regions are interconnected with progresses. MRI/VBM studies on brains of living AD-
each other [14], causing some investigators to describe affected humans and postmortem brains of humans
AD as a connection syndrome and others as a dis- who died with AD have confirmed this observation
connection syndrome. Olfactory pathway-innervated and enabled a consensus view on the general sequence
brain regions include certain neocortical regions. of AD progression.
Subcortical neurons that directly project to these neo- The entorhinal cortex and the transentorhinal cortex
cortical regions are also selectively vulnerable in AD. are damaged earlier in AD than other limbic regions.
It has been postulated that AD neuropathology results Damage in the limbic regions is followed by NFT
from an agent that crosses synapses, spreading in an damage in temporoparietal neocortices with associ-
anterograde direction between connected corticocorti- ation areas affected earlier and to a greater extent
cal and corticosubcortical cells of AD-vulnerable brain than primary sensory areas. The frontal and occipi-
regions [26]. Spread of an agent from one brain region tal cortices then show damage while the sensorimotor
to another may provoke cell changes that lead to the cortices are largely spared. Some subcortical regions
formation of NFTs in the soma and dendrites of tar- are also involved in AD but those of the brainstem
get neurons. For example, hyperphosphorylated tau have been less studied. AD-affected brain regions are
has been proposed to spread between interconnected all interconnected.
brain regions in AD [35–37]. An alternate explanation AD preferentially affects certain cell types in AD-
for the apparent spread of hyperphosphorylated tau in vulnerable brain regions. Next we consider possible
interconnected brain regions is discussed below in the reasons why certain types of neurons are preferentially
section “Aluminum and AD Progression”. affected, both earlier and to a more severe extent than
AD damage may also spread between specific brain other cell types.
regions in a retrograde direction since synaptic termi-
nals have active incorporation of plasma membrane,
indiscriminately taking in neuropil substances that CELLULAR AND TISSUE FEATURES THAT
can be transported back to the cell soma [38]. This INCREASE CELL SUSCEPTIBILITY TO
mechanism involves plasma membrane invagination NFT FORMATION
with vesicles budding into the cytoplasm [39]. Small
membrane-bound vesicles are transported retrogradely The large corticortical stellate neurons in layer II
by kinesin along microtubules at rates similar to fast and underlying pyramidal cells in the superficial part of
anterograde transport [40]. AD progression could also layer III of the entorhinal cortex are, as mentioned, the
spread simultaneously in both anterograde and retro- cells of origin for the perforant pathway (Fig. 1) [25].
grade directions via different sets of fibers [14]. These particular cells have a high risk for developing
Paradoxically, the neuropathology of AD is also NFTs in AD and must have at least one, or more, unique
consistent with its being a disconnection syndrome, qualities that predispose them to NFT formation.
involving disruption to afferent and efferent connec- Several cellular and tissue features have been pro-
tions of NFT-containing corticocortical cells [27]. AD posed to account for this phenomenon.
neuropathology probably involves connections at one
stage and disconnections at another. For example, cell- • Firstly, the cell types that develop NFTs are gen-
cell connectivity could allow the propagation of a erally complex with a long axon and large surface
neurotoxic agent throughout a sequence of specific area [42].
brain regions, giving rise to NFTs and cytoskeletal • Secondly, they have high energy demands, high
damage that eventually disrupt these connections and densities of transferrin receptors, and elevated
result in functional loss. iron uptake [43, 44].
inhibitory interneurons seldom, if ever, contain NFTs

[46].
The total dendritic length in layer II neurons of the
entorhinal cortex and the hippocampal CA1 zone of
non-human primates can both reach 8.0 mm, implying
these two cell types have comparable complexity [42].
However, the entorhinal cortex is consistently dam-
aged earlier and more severely by NFT formation than
the CA1/subiculum zone despite the comparable com-
plexity of corticocortical cells in both brain regions.
This led Stranahan and Mattson [42] to conclude that
some other cell feature must also be important.
High energy demands, densities of transferrin (Tf)

receptors, and iron uptake
A second possibility for increased cell vulnerabil-

ity to AD could come from particularly high energy
demands, high densities of Tf receptors, and the
large mitochondrial iron requirement necessary to gen-
erate sufficient ATP to meet such high metabolic
Fig. 1. Entorhinal cortex cells of origin for the perforant pathway. requirements. Cells with these features may be more
Schematic representation of the perforant pathway. The perforant vulnerable to NFT formation than cells with relatively
pathway is similar for humans and rats apart from minor variations.
low metabolic demands.
The cells of origin (CO) for the perforant pathway reside in layer
II (shown as cell islands) and in the superficial part of layer III of Plasma iron circulates in the form of transferrin-
the entorhinal cortex (EC). The cells of origin receive information bound iron (Tf-iron). Figure 2 illustrates iron uptake
from many cortical regions. Axons of the cells of origin converge and utilization, storage, and exit from a generic cell
in the angular bundle (AB). The axons leave the angular bundle and
diverge into fascicles known as the perforant pathway (PP) because
[47]. These processes are similar in capillary endothe-
the axons perforate through the gray matter of the subicular cortex lial cells of the blood-brain barrier. The Tf-iron attaches
(SC) to the hippocampal formation (dentate gyrus and hippocam- to Tf receptors on the luminal surface of the cerebral
pus proper). A contingent of fascicles enters the stratum lacunosum capillary endothelium, initiating iron transport across
moleculare (SLM) of the hippocampal CA1/subicular zone (CA1).
More fascicles cross the hippocampal fissure (HF) to enter the molec-
the blood-brain barrier [49]. Upon being released to
ular layer (ML) of the dentate gyrus (DG). Reproduced from [41] the cytoplasm of endothelial cells, iron excessive to
(Copyright: JR Walton). that required by the endothelial cell is transported
across its abluminal surface and into the extracellular
• Thirdly, NFTs form more readily in cells that are matrix by ferroportin. Iron also crosses the blood-brain
poorly myelinated [45]. barrier by Tf-independent mechanisms [50, 51]. Iron
• Fourthly, a cell may be more susceptible to NFT exiting from the endothelium binds to extracellular Tf
formation if exceptionally well-vascularized. generated by oligodendrocytes, astrocytes, and/or the
choroid plexus [52]. The Tf-bound iron is then taken up
into neurons via Tf receptors on the neuronal surface
Cell size and complexity [53].
The reasoning behind the proposal that complex High energy demands
cells with a large surface area are most prone to Multipolar stellate cells and pyramidal cells in
NFT development is that these traits would provide islands of the superficial entorhinal cortex are espe-
increased exposure to toxic agents in the extracellu- cially vulnerable to NFT formation. These cells of
lar milieu [42]. This proposal is probably based on origin for the perforant pathway are grouped into func-
observations that most cells that develop NFTs are tional modules that have particularly heavy metabolic
large corticocortical and corticosubcortical pyramidal demands during waking hours and paradoxical (REM)
or stellate cells. In contrast, small neurons, such as the sleep [54]. Cytochrome oxidase staining confirms that
spiny stellate cells in entorhinal cortex layer IV and these entorhinal cortical cells function as cytochrome
distribution to those with neurons that have with high

numbers of mitochondrial respiratory chains and high
cytochrome oxidase levels [59]. Cytochrome oxidase,
the terminal enzyme of the electron transport chain,
serves as a marker of metabolic activity in neurons
[44].
The entorhinal cortex, hippocampus, amygdala,
temporal and parietal cortex, locus coeruleus, and dor-
sal raphe nucleus all contain relatively high densities of
Tf receptors compared to other brain regions [43, 44,
59–61]. However, high Tf receptor density on its own is
insufficient to explain selective neuronal vulnerability
to AD since other cell groups that have high Tf recep-
tor densities, such as the olives and trigeminal nucleus,
fail to show evidence of selective neurodegeneration in
AD [60]. This suggests that high Tf receptor density in
cells is more relevant to AD vulnerability if those cells
are interconnected with cells in other AD-vulnerable
brain regions.
Iron regulatory proteins (IRPs)

Fig. 2. Generic diagram of iron uptake into cells. A) Tf-iron binds Iron is an essential metal in cell metabolism for ATP
to Tf receptors (TFR) at the cell surface. B) The Tf-iron/TfR com- production, DNA polymerases and repair, synthesis of
plexes are internalized in clathrin-coated vesicles. C) ATP-dependent
proton pumps (H+ ) acidify the endosome, releasing iron from the
heme proteins, and catecholamine neurotransmitters.
Tf/TFR complex. D) Iron exits the endosome via the divalent metal Each mitochondrial respiratory chain requires up to
ion transporter DCT1 [48]. E) The free iron (Fe2+ ) is either used 40 iron atoms [61]. Intracellular iron metabolism is
by the cell for mitochondrial respiration or incorporated into iron- controlled by iron regulatory proteins 1 and 2 (IRP1,
containing proteins. F) The remainder of iron is either stored in
ferritin deposits or transported out of the cell via ferroportin (not IRP2), that act as iron sensors, as described in Fig. 3
shown). G) Meanwhile, the Tf/TFR complex is returned to the cell [62, 63]. Normally, when cells are iron-deficient, syn-
membrane where the complex dissociates, releasing Tf for re-use. thesis of the iron-storage protein ferritin is repressed
Reproduced from [47] with permission from John Wiley & Sons. (Fig. 3A) whereas Tf receptor synthesis and iron uptake
resume (Fig. 3B). When the cells have enough iron,
ferritin synthesis is activated (Fig. 3C) and Tf receptor
oxidase-rich modules [54]. Intrinsic electrical prop- synthesis shuts down, reducing iron uptake (Fig. 3D).
erties of the stellate cells, involving oscillations This process is cyclical in healthy cells, ensuring that
associated with gamma and theta rhythms [55], are cells have sufficient, but not excessive, free iron.
highly correlated in all behavioral states and also Iron metabolism becomes disregulated in the most
contribute toward their necessarily high bioenergetic vulnerable regions of AD brains. The disregulation
needs [54]. Cells in the perirhinal cortex (including the occurs as a result of IRP2 stabilization which in turn
transentorhinal cortex) also display oscillations in the maintains Tf receptor mRNA in a stabilized state [65].
theta range [56]. Consequently, corticocortical cells in IRP2 stabilization makes the cells behave as though
both the entorhinal and perirhinal regions have high they are in a permanent state of iron deficiency. This
glucose utilization and a very high level of mitochon- stabilized state occurs because, for some reason, IRP2
drial respiration [57]. does not degrade as normally happens when iron levels
are adequate. Consequently, cells in these vulnerable
Transferrin receptor density regions continue to synthesize Tf receptors and import
Neurons with large metabolic demands have high more iron than the cell ferritin deposits can store.
densities of Tf receptors on their surface that allow In AD, iron levels become significantly more ele-
them to import sufficient iron to meet their high-energy vated in the hippocampus, amygdala, temporal cortex,
demands [43]. Glial cell membranes also have Tf and parietal cortex compared to control brains [66].
receptors [58]. Brain regions containing neurons with This excess iron contributes to increasing oxida-
high densities of Tf receptors show almost identical tive damage in large corticocortical cells of the
Fig. 3. Iron regulatory proteins (IRP) 1 and 2 under low and high iron conditions in neurons. Ferritin mRNA activity (A&C) is reciprocal with
Tf receptor mRNA activity (B and D, respectively). A) When intracellular iron is deficient, IRPs 1 and 2 bind with high affinity to the iron
regulatory element (IRE) in the 5 UTR of ferritin mRNA, repressing its translation. B) IRP binding to the five IREs in the 3 UTR of Tf receptor
mRNA stabilize its translation by protecting the mRNA from RNase degradation. This allows the continuation of Tf receptor synthesis and
iron uptake until the iron supply is again replete. C) The small spheres indicate excess ferric iron ions (Fe3+ ). When the iron supply is replete,
Fe3+ binds to a Fe3+ -binding site on the IRP2, oxidizes the IRP2 and causes it to lose its affinity for the IRE. IRP detachment from the 5 end
of ferritin mRNA activates ferritin translation. The dotted line indicates a deteriorating IRP2 that has detached from its IRE and signaled for
degradation by the ubiquinol-proteosomal pathway (61–63). The higher Fe3+ level changes the conformation of IRP1 from an RNA-binding
protein to an inactive form that binds an iron-sulfur cluster (4Fe-4S). D) As in C, Fe3+ binds to iron-binding sites on the IRP2s, oxidizing them,
and causing IRP2 detachment from the IREs and signaling for IRP2 degradation [62–64]. This exposes the Tf receptor mRNA to degradation
by intracellular RNases. Consequently, iron uptake ceases. Reproduced from [61] with permission from Elsevier.
AD-vulnerable brain regions. Excess iron imported entorhinal cortex, hippocampal formation, and amyg-
into these neurons participates in NFT formation by dala are in the phylogenetically-oldest allocortex and
aggregating hyperphosphorylated tau and binding to these are the most poorly myelinated and most NFT-
NFTs [67]. prone regions of the AD brain [45].
Increased vulnerability is conferred on the poorly
Scanty myelination increases cell susceptibility to myelinated entorhinal cortical neurons by their huge
NFT formation demands for energy generation. A high degree of
axonal myelination on neurons in primary sensory
Poorly myelinated cells are highly prone to NFT for- areas increases the velocity of their signal conduc-
mation [68, 69]. Primary sensory areas of the neocortex tion while reducing cellular energy expenditures about
are very well myelinated but the density of myelina- 5,000 times [71]. Conversely, poorly myelinated neu-
tion decreases with increasing distance from primary rons, such as those of the entorhinal cortex, have
sensory areas [68–70]. Thus, neocortical association leaky axons and must generate vastly (∼5,000-fold)
areas are less well myelinated, particularly in the higher more energy to compensate for the significant wastage.
order association areas. Limbic tissues including the Poorly myelinated pyramidal cells are much more
susceptible to oxidative damage. Consequently, those

cells accumulate lipofuscin pigment and exhibit early
evidence of cellular aging [45].
Poor myelination in the olfactory limbic system is
likely to be one reason why the entorhinal cortex, hip-
pocampus, amygdala, and olfactory bulb are generally
more vulnerable to NFT pathology than neocortical
regions. However, poor myelination on its own fails to
explain why the entorhinal cortex is more vulnerable
to NFT formation at an earlier time than other limbic
structures.
Redundant vascularization may increase cell

susceptibility to NFT formation
The final feature considered here that may increase a

cell’s susceptibility to NFTs pertains to vascular redun-
dancy. Neuronal firing increases blood flow by evoking
the redistribution of patent capillaries [72]. Hence,
neurons with high firing activity increase blood flow.
Conversely, neurons damaged and unable to fire have
a decreased vascular supply.
Blood flow to a brain region is also dependent on
capillary density. This may explain why stellate and
pyramidal cells in the superficial entorhinal cortex are
selectively more vulnerable to NFT pathology than
cells in other limbic regions. The entorhinal cortex of Fig. 4. Vascularization of cell islands in the human entorhinal cortex.
the guinea pig brain is supplied by caudal and rostral A) This tangential section through layer II of the human entorhinal
posterior arteries, with a very broad overlap between cortex is stained with cresyl violet. Note the islands of large neurons.
their distal territories, and also by the middle cerebral B) An adjacent section to that shown in A, stained with the Gallyas
method, shows the complex axonal matrix that surrounds the cell
artery [73]. Such redundancy is potentially protective islands. Note the satellite arterioles (e.g., arrow) among the surround-
against hypoxic events. The redundant vascular input ing axonal fibers. The arterioles that surround the cell islands branch
to the entorhinal cortex also increases its exposure and give rise to a dense capillary meshwork that heavily invests the
cell islands. Reproduced from [1] with permission from John Wiley
to blood-borne toxicants and inflammatory factors. In
& Sons.
contrast, the ventral part of the hippocampal forma-
tion is exclusively supplied by the rostral posterior
cerebral artery [73]. This may explain hippocampal rier into the extracellular matrix of well-vascularized
susceptibility to transient global ischemia [74]. neurons.
The human entorhinal cortex receives its vascular
input from both the posterior and middle cerebral arter- LARGE CELL SIZE, HIGH ENERGY AND
ies and has abundant anastomotic links [75]. Satellite IRON DEMANDS, SCANTY MYELINATION,
arterioles, among the dense reticular matrix of axons AND REDUNDANT VASCULARIZATION
that surround islands of multipolar stellate cells and ARE INSUFFICIENT TO CAUSE NFT
pyramidal cells in layer II, give rise to a dense mesh- FORMATION
work of capillaries that invade these islands (Fig. 4)
[1]. Here we focus on the large corticocortical cells
Capillaries of different brain regions have a rela- of the superficial entorhinal cortex that become pro-
tively uniform density of Tf receptors on their surface foundly affected at a relatively early stage of AD [77],
[76]. However, a higher capillary density should pro- to assess whether they have any single feature, or
vide more endothelial cells and more Tf receptors that combination of the cell and tissue features described
can, in principle, move larger amounts of iron and above, that might account for their susceptibility for
other blood-borne agents across the blood-brain bar- early NFT formation. Stellate and pyramidal cells of
the superficial entorhinal cortex are poorly myelinated ples from 52 autopsy-confirmed AD patients [80],
cells, extremely active, and have a high density of Tf transplanting them into the brains of 61 non-human
receptors on their surfaces and high iron uptake. The primates to learn whether a transmissible agent is
superficial entorhinal cortex has a redundant vascular involved in AD. Only two animals developed a spongi-
supply and its cell islands are surrounded by a dense form encephalopathy. A further 17 cases were tested
capillary network. for more than 50 months without any effect. The
It is likely that each of the proposed features con- investigators re-tested samples from the same brain
tributes to increased susceptibility for NFT formation tissue that had previously produced positive results for
in these cells of origin for the perforant pathway. encephalopathy. Positive findings were elusive upon
However, these features are also characteristic of their re-testing. This led the authors to conclude that an
counterpart cells that are functioning well in healthy infectious agent is not involved in AD causality [80].
young and non-demented aged brains. Hence, a trigger Subsequent testing of 36 additional AD brain samples
that disrupts normal cell metabolism in these AD- confirmed these conclusions [81].
vulnerable cells is also necessary. These AD results contrast with those from another
NIH study of prion disease transmissibility. Trans-
AGENTS PROPOSED AS THE planted brain samples into non-human primates
ENVIRONMENTAL COMPONENT OF AD showed transmission rates of 100% for eight subjects
with iatrogenic Creutzfeldt-Jakob disease, 90% for 234
The main candidates for the environmental com- patients with sporadic Creutzfeldt-Jakob disease, 68%
ponent of AD are prions (atypical slow viruses), for 36 cases with familial Creutzfeldt-Jacob disease,
conventional viruses, and metal neurotoxicants. These and 95% for 18 (biopsied) patients with kuru [81].
candidates are reviewed to determine which best fits the These samples were injected into 45, 1167, 197, and
profile of an environmental trigger that slowly accumu- 45 primates, respectively. As for AD, animals that died
lates in the brain over decades, specifically in affected from any cause were given neuropathological exam-
neurons of AD-vulnerable brain regions, to reach toxic inations and designated as positive if they showed
levels in old age and produce AD neuropathology with- typical spongiform change in their brain.
out rapidly lysing the affected neurons.
A microorganism proposed as the environmental Common viruses
cause of AD should fulfill Koch’s postulates. These
are: (1) the microorganism must be present in every Common viruses have also been considered as possi-
case of the disease under study; (2) the microorgan- ble environmental causes of AD. Herpes simplex virus
ism should be capable of being isolated and grown in 1 (HSV1) can persist in a latent form for many years
pure culture; (3) the microorganism must, when inocu- without showing overt disease after primary infec-
lated into susceptible animals, cause the disease under tion. It can reactivate from the latent state and cause
study; (4) The microorganism must be recovered from cold sores and mouth, throat, face, and eye problems,
the inoculated animals and identified. despite the presence of circulating antibodies. HSV1
is one of many micro-organisms that stimulate NF␬B
Prions [82] which in turn upregulates the gene for A␤PP [83],
and genes for other stress-response proteins, leading to
Human prion diseases are infectious, transmissible, increased A␤ formation in experimental systems [84].
and hereditable, between species as well as within HSV/HSV1 immunoreactivity has been reported in
species unlike AD. The brain regions that are affected AD and normal brain tissue [85–87] and HSV DNA
relatively early in prion diseases include the pulvinar, was detected in 3 of 4 brain smears [88]. However,
ventrolateral, and mediodorsal thalamic nuclei, the larger DNA hybridization studies have been unable to
putamen, and caudate nucleus [78]. The hippocampal confirm the specific presence of HSV DNA in AD brain
formation is relatively spared. Damage in these brain tissue [89–91]. HSV antibody titers of AD patients are
regions is inconsistent with AD progression. Prion similar to those of controls [92–94].
protein deposits, vacuoles, and rapid cell death fur- HSV1 is also neurotrophic. HSV1 can produce Her-
ther distinguish prion disease neuropathology from AD pes simplex encephalitis (HSE) and may be the only
neuropathology [79]. microorganism that specifically attacks the stellate and
A large study conducted at the National Institutes pyramidal cells of origin for the perforant pathway,
of Health carried out extensive testing of brain sam- producing an encephalitis with AD-like impairment of
short-term memory [95, 96]. Then again, HSE is a rare a toxic agent in the brain over decades of time [26,
condition that affects children and adolescents as well 83]. If aluminum is indeed causal to AD, its pro-
as older people, with 50% of HSE cases being under longed accumulation is likely to be life-long, with
age 50 [97]. Unlike sporadic AD, HSE shows no prefer- aluminum exposure beginning at, or even prior to, birth
ence for the elderly. Abundant immunoreactivity was [105–107].
observed in temporal lobe regions of an encephalitis
case. Large numbers of glia and neurons showed evi- ACUTE, SUB-ACUTE, AND CHRONIC
dence of active infection. Similar HSV antigenicity is FORMS OF ALUMINUM NEUROTOXICITY
not observed in any temporal lobe region of AD cases
or controls [98]. Approximately 70% of untreated HSE There are three main forms of aluminum toxicity
cases undergo rapid death [97] and 2.5% regain normal (acute, sub-acute, and chronic). All three forms share
brain function. Relapses of infections can occur within some similarity and yet are significantly different from
weeks or months [99]. These traits are uncharacteristic each other. They are similar is that they all cause some
of AD. form of brain disease. The three forms differ in the type
Proponents for HSV1 causality of AD describe a of subjects they affect. The rates at which these condi-
scenario in which HSV1 enters the brain in old age [84]. tions occur and the aluminum concentrations involved
This late and rapid progression is inconsistent with are distinctly different.
the slow progression of AD, in which brain changes
gradually give rise to MCI and then to overt AD [100]. Acute aluminum neurotoxicity
In summary, large AD transplantation studies car-
ried out at the NIH failed to show evidence of Acute aluminum neurotoxicity occurs when a large
transmissibility [80, 81]. Also, serum antibody titers amount of aluminum (as much as 500 ␮g/l or more)
measured in AD patients and non-demented controls enters the circulation. Acute aluminum neurotoxic-
for HSV, and other common viruses (Measles virus, ity can affect humans with normal kidney function as
Adenovirus, Coxiella burnettii, Cytomegalovirus, well as those with chronic renal failure, resulting in
Influenza A, Influenza B, Chlamydia Group B (psit- an encephalopathy that typically involves grand mal
tacosis virus), and Mycoplasma pneumoniae) have all seizures, culminating in coma and death within days
failed to show statistically significant difference [93, or several weeks [108, 109].
94]. Overall, the results indicate that, in the case of
AD, Koch’s postulates have yet to be fulfilled by any Sub-acute aluminum neurotoxicity
infectious agent.
Sub-acute aluminum neurotoxicity involves inter-
Toxic metals mediate aluminum levels in blood or CSF over
several years. An example is dialysis encephalopathy
Metal neurotoxicants have been suggested as envi- syndrome (DES) or dialysis dementia, a progres-
ronmental candidates for AD etiopathogenesis. Lead sive encephalopathy that particularly affects dialysis
neurotoxicity preferentially affects children up to 4 patients with chronic renal insufficiency routinely
years of age. One survivor of lead encephalopathy, who exposed to high levels of circulating aluminum
died at age 42 years with severe mental deterioration, [110]. Dialysis patients have impaired ability to
had brain lead levels ten times higher than those of excrete the aluminum they acquire, mainly from
AD patients [101]. Nevertheless, the evidence for lead aluminum-containing products such as phosphate
involvement in AD is scant. binders, antacids, and/or contaminant aluminum in
Blood mercury levels were found to be twice as high dialysis water. DES onset is insidious, often involv-
in AD patients as in controls [102]. Most evidence ing problems with speech. Seven to nine months after
relating to brain mercury levels in AD and human symptoms first appear, the patient becomes totally
controls indicates that mercury does not progressively mute, unable to perform purposeful movements and
accumulate in human brains with increasing age as soon dies.
does aluminum [103, 104]. Aluminum best matches DES epidemics have previously occurred in dialy-
the profile for the AD environmental candidate. sis patients [110–112]. Removal of aluminum from the
Old age is commonly regarded as a major risk fac- dialysis water and dialysis equipment, monitoring of
tor for AD. AD’s long prodromal phase suggests that plasma aluminum levels, and interventional treatments
AD involves the slow and prolonged accumulation of with aluminum-chelating agents, usually desferriox-
amine, have prevented many adult deaths from this for significant NFT formation unless treated by kidney
cause [113]. In general, DES and dialysis patients tend transplantation.
to be younger than most sporadic AD patients. NFTs also form very slowly in brains of non-human
In a study by Harrington et al. [114], the mean primates, even after aluminum injection directly into
brain aluminum content was 7.35 ␮g/g in dialysis the brain [116]. In contrast, aluminum-induced NFTs
patients compared to 1.95 ␮g/g in controls. Brain develop in brains of rabbits and cats within 48 and
aluminum content correlated with plasma aluminum 76 hours, respectively, after intracerebral aluminum
content (r = 0.772, p = 0.008) and with terminally trun- injection [117, 118], showing species difference in the
cated tau protein in white matter (r = 0.753, p = 0.001). timing of NFT formation.
Eight of 15 brains from dialysis patients exhibited
A␤ immunoreactivity. Electron microscopy revealed Chronic aluminum neurotoxicity
twisted filaments, indistinguishable from those in
NFTs of AD brains, in two brains of dialysis patients Finally, there is chronic aluminum neurotoxicity.
with the highest levels of hyperphosphorylated tau in Considerable evidence supports the possibility that
their gray matter [114]. The observed frequency for AD is a form of chronic aluminum neurotoxicity that
these AD-type changes exceeded the expected fre- occurs in humans.
quency for AD changes in this group (38–68 years;
0–1%, p < 0.0001), corresponding to the expected fre- ALUMINUM EXPOSURE AND
quency for AD only above age 80. ABSORPTION OF INGESTED ALUMINUM
Five of the six brains from dialysis patients with
high aluminum showed depletion of normal tau accom- The main source of aluminum exposure for most
panied by accumulation of hyperphosphorylated tau humans is from oral ingestion of aluminum additives
protein [114]. This phenomenon of progressive nor- used to enhance various aspects of commercially-
mal tau conversion to hyperphosphorylated tau also prepared foods and beverages, including alum-treated
occurs in AD [115]. The tau-related changes reported drinking water [119]. In water treatment parlance, alum
in brains of these dialysis patients are comparable to generally refers to aluminum sulfate (Al3 (SO4 )3 ), a
those that occur in AD brains at an earlier stage and relatively soluble form of aluminum used in the clarifi-
that eventually lead to NFT formation. cation of some bottled waters and many urban drinking
Some people feel that brains of dialysis patients with water supplies.
high plasma aluminum levels would have abundant Aluminum compounds have versatile properties and
NFTs if aluminum is involved in NFT formation in serve many useful functions as additives: anti-caking
AD patients. However, given that chronic renal fail- agents in salt, coffee whitener, pancake mix, and other
ure and AD are two distinctly different disease entities powdered foods; emulsifiers and melting agents in
that develop at vastly different rates, their neuropathol- cheeses; clarifying agents in water, puddings, and other
ogy can also be expected to differ. DES patients have processed foods where precipitates may form; pickling
very high brain aluminum concentrations (e.g., 25 ␮g/g agents; meat binders for sausages and luncheon meats;
brain tissue (dry weight)). This may result in aluminum hardening agents for candied fruits; gravy and sauce
precipitates, forming deposits in lysosomes of cells thickeners; rising agents in baking powder, self-rising
instead of reacting with hyperphosphorylated tau to flour, and baked goods; dough strengtheners; buffering
form NFTs as aluminum does when present in plasma and neutralizing agents; and mordants that bind dyes
and brain concentrations 2–3 times higher than normal to confectioneries to make them colorful.
values. Aluminum absorption occurs across all parts of the
NFT paucity in brains of dialysis patients and DES intestine including the colon [120]. A major reason
patients is also likely to relate to: (1) the age difference why aluminum accumulates so slowly in the brain
at which most patients develop chronic renal failure is because most ingested aluminum is blocked from
or sporadic AD; (2) the sub-acute aluminum exposure absorption into the systemic circulation by a mucus
period over which plasma levels in chronic renal fail- layer that lines the gastrointestinal tract [121].
ure patients are significantly elevated (amounting to a Small amounts of Al26 , a radioactive tracer for nat-
few years rather than four or more decades); and (3) the ural aluminum (Al27 ), are detectable by accelerator
extremely slow, decades-long time over which human mass spectrometry in the brains of rats within weeks
NFTs are estimated to form [22]. Dialysis patients after ingesting an equivalent amount of Al26 to the
may die of their condition prior to the time required aluminum contained in a single glass of alum-treated
drinking water [122–124]. Al26 is not found in nature Aluminum is a light element present in the brain
so its presence in the brain after 26 Al treatment is in small quantities. Some instrumental techniques,
unambiguous. Non-dietary sources of aluminum fur- including nuclear microscopy, X-ray fluorescence,
ther contribute to the aluminum burden of the brain and scanning electron microscopy with energy-dispersive
skeletal tissues. Trans-dermal aluminum absorption X-ray analysis (except in NFTs, where aluminum is
occurs from topical applications (mainly aluminum- more concentrated), and flame atomic emission meth-
based deodorants and sunscreens) [125, 126]. Some ods are too insensitive to measure trace levels of
injected vaccines include aluminum as an adjuvant to aluminum in biological samples over the 1 to 7 parts
boost the body’s immune response [127, 128]. Simu- aluminum per million (ppm) range contained in AD-
lated vaccination in mice produces an aluminum peak vulnerable brain regions. Also, certain other elements,
in their brains 2–3 days post-injection [129]. Alu- mainly magnesium and phosphorus, can interfere with
minum is a component of some pharmaceuticals (such aluminum detection unless appropriate measures are
as aluminum antacids and buffered aspirins) [130] and taken to prevent this problem. Aluminum measure-
certain medical treatments such as irrigation of the ments require clean-room conditions.
bladder [131]. Aluminum is also contained in a bone
cement used in surgery [132]. Additional information
THE DILEMMA IN PERFORMING
about human exposure to aluminum is available else-
EPIDEMIOLOGICAL STUDIES THAT
where [133, 134].
ASSESS THE EFFECTS OF CHRONIC
More aluminum enters than leaves the brain, result-
HUMAN EXPOSURE TO A NEUROTOXIN
ing in a net accumulation of aluminum in the brain
over time. Aluminum is the only common neurotoxi-
The dilemma is that a properly-designed epidemio-
cant known to accumulate in the brain with increasing
logical study capable of providing the highest level of
age, even in non-demented humans albeit at lower rates
evidence that AD is a human form of chronic aluminum
[135–138]. In 2007, the UN/WHO expert commit-
neurotoxicity would be unethical to perform. It would
tee on food additives reduced the provisional weekly
also be impossible to obtain cohorts of human subjects
tolerable level for aluminum from 7 mg Al/kg body-
who could comply with a prospective, decades-long,
weight (bw)/week to 1 mg Al/kg bw/week [139]. Many
randomized controlled trial involving assignment to
humans routinely consume considerably more alu-
treatment groups that consume either high, medium,
minum than this recommended limit [133].
or low concentrations of aluminum in their food and
water, over several decades, to learn whether chronic
METHODS THAT ALLOW POSTMORTEM aluminum ingestion results in AD. In lieu of any exist-
ALUMINUM DETECTION IN BRAINS OF ing human study of this nature, the author applied a
HUMANS AND EXPERIMENTAL ANIMALS comparable protocol to an animal population.
Aluminum can be visualized postmortem in hip- Rodents that mimic human dietary aluminum
pocampal and cerebral neurons of the AD brain exposure
[140] with at least three different staining tech-
niques: the morin fluorescent stain [141], Walton bright Chronic aluminum neurotoxicity is best studied
field/fluorescent stain [142], a modified protocol for using human-equivalent aluminum exposure in short-
the Walton stain [143], and an immunostain against lived laboratory animals over most of their lifespan
protein-bound aluminum [144, 145]. [41, 143, 146]. A progressive dementia-like condition
A variety of instrumentation is also available develops in a dose-dependent manner in rats that con-
for aluminum detection or measurement. These sume soluble aluminum in their drinking water for
include graphite furnace atomic absorption spec- most of their adult life, in total amounts equivalent
trometry, inductively-coupled plasma atomic emission to those consumed by humans living in contemporary
spectrometry, laser microprobe mass analysis, scan- westernized societies from their foods, beverages, and
ning/transmission electron microscopy and transmis- aluminum additives [133, 134]. This approach mimics
sion electron microscopy with energy-dispersive X-ray the aluminum dietary levels consumed by humans as
analysis, secondary ion mass spectrometry (SIMS), well as the prolonged duration of human exposure to
and accelerator mass spectrometry used in conjunction dietary aluminum. As it turned out, this animal model
with the 26 Al radioisotope. developed cognitive deterioration in old age after a long
Fig. 5. Line and scatter plots, with Loess smoothing, exemplify overall T-maze performance scores of rats from the three aluminum treatment
groups. Each dot recognizes one weekly test score for choice accuracy out of 100%. A) Scores of a rat that chronically consumed a low level of
aluminum in the human dietary aluminum range and remained cognitively-intact. B) Scores of a rat that exhibited cognitive deterioration after
chronically consuming aluminum at an intermediate level. C) Scores of a rat with cognitive deterioration that chronically consumed aluminum
equivalent to an amount at the high end of the human total dietary aluminum range. Reproduced from [145] with permission from Elsevier.
prodromal period and proved to be a faithful transla- lower mean T-maze performance scores in their old
tional rat model for AD [41, 143]. age (Fig. 5) than in their middle age and developed
Aluminum treatment was delayed until the rats were progressive cognitive deterioration from around ages
at age 12 months (commencing their middle age) to 27–28 months. Their mean lifespan was 30 months of
allow time for normal brain development. All animals age.
in this longitudinal study were exposed to the same Testing continued until a terminal condition became
levels of aluminum in the air they breathed and in the evident. The longest-living animals were tested on
measured food aliquots they received. The amounts of more than 100 occasions. The rats that developed cog-
water the rats consumed ad libitum were also mea- nitive deterioration in old age showed no evidence
sured. The only treatment difference between the three of improvement with continued testing, indicating
rat groups was the amount of aluminum contained in that their condition was irreversible. These rats dis-
their drinking water [146]. This level of controlled played abnormal neurological signs and exhibited
dietary aluminum exposure is more accurate than could novel behaviors such as confusion, attentional deficit,
be achieved with a human population. perseverative activity and urinary incontinence while
in the T-maze. The rats that developed cognitive
Training and weekly testing on a task used for deterioration in old age did so in an aluminum dose-
memory assessment dependent manner. Almost all rats in the study had
normal kidney and liver functions in old age [146].
The rats were trained to perform a rewarded con- Rats that developed chronic aluminum neurotoxic-
tinuous alternation T-maze task commonly used for ity/cognitive deterioration generally did so at a mean
memory assessment [147]. Rats that died before 28 age of age 28 months, after 16 months of chronic alu-
months of age, or were unable to perform all ten choices minum exposure at human relevant levels. Wistar rats
of the T-maze task within 5 minutes, were excluded are estimated to age approximately 35 times faster than
from the study. The study rats were tested on the T- humans [148] so 28 month old rats would approximate
maze task each week from age 9 months to the end of the age of 82-year old humans. The 16 month expo-
their natural lifespan. They performed the maze task sure period is almost equivalent to a human exposed to
with 70%–100% accuracy upon entering middle age dietary aluminum for 47 years.
(at 12 months). The amounts of aluminum the animals consumed
All rats that consumed aluminum at the low end of correlated positively with their serum aluminum lev-
the human dietary aluminum range performed the task els. Animals in the highest aluminum dose group had
in old age as well as, or better than, they had performed a mean serum aluminum level that was twice as high
in middle age. Twenty percent of the rats that con- as that of the low aluminum controls yet some rats in
sumed aluminum at the intermediate dose level and the highest aluminum group had serum aluminum lev-
seventy percent of those that consumed aluminum at els that were six times higher than the low aluminum
the high dose level, that is, at the high end of the human controls. Two rats in the intermediate group had higher
total dietary aluminum range, obtained significantly serum aluminum levels than other members of their
accumulation compared to 23% at this stage in low

aluminum controls [143]. These are the cells of origin
for the perforant pathway (Fig. 6).
Also, high-stage nuclear aluminum accumulation
affected a much larger proportion of the superficial
entorhinal cortical cells than of cells in any other brain
region examined. Cells in the hippocampal subicu-
lum/CA1 zone tended to exhibit high stage aluminum
accumulation in the form of discrete cell bands or
lesions.
The temporal association cortex of rats with cog-
nitive deterioration also had a significantly larger
proportion (40%) of neurons with high-stage alu-
minum accumulation than in the same brain region
of controls (13%) [143]. The temporal association
cortex of cognitively-deteriorated rats also exhib-
ited many more pyramidal cells with high stage
aluminum accumulation than the primary sensory cor-
tex. High-stage aluminum in the nucleus of the rats
with cognitive deterioration preferentially affected the
amygdala, olfactory bulb, piriform cortex, frontal cor-
tex, nucleus basalis of Meynert, dorsal raphe nucleus,
locus coeruleus, and other brain regions vulnerable to
NFT damage in AD [146].
Fig. 6. Staining for aluminum in the entorhinal cortex of a The brain regions most affected in this aluminum-
cognitively-intact rat (A) and a rat with cognitive deterioration (B).
inducible translational rat model are homologous to
(A) Stellate and pyramidal cells of origin for the perforant pathway
from a low aluminum control stain blue, lacking the magenta staining those most affected in humans with AD [13, 146].
characteristic of aluminum with the Walton stain. Aluminum stain- Cognitive deterioration in these rats involves destruc-
ing in this section primarily stains glial cells and erythrocytes. (B) In tion of the perforant pathway by a mechanism shared
contrast, stellate and pyramidal cells of origin for the perforant path-
way in the entorhinal cortex, from a rat with cognitive deterioration,
by humans with AD [1, 29, 41]. In an earlier study,
stain magenta to purple, indicating high-stage aluminum accumula- aluminum was directly injected into the brains of rab-
tion. Reproduced from [149] with permission from the Royal Society bits and NFT damage was observed in regions of the
of Chemistry. rabbit brains homologous to those where NFTs form
in AD brains [150]. Other studies reported that dialy-
sis patients exhibit aluminum deposition in the same
treatment group. Those two rats were the only ones in brain regions that are vulnerable to NFTs in AD [43,
their group to develop cognitive deterioration in old 44, 151]. Thus, several models have shown that alu-
age, indicating they absorbed more aluminum than the minum preferentially accumulates in brain regions that
others that consumed the same aluminum dose level. are particularly susceptible to damage in AD.
This may reflect some difference in their genetic con-
stitution that enhances aluminum absorption [146].
AD PATIENTS ABSORB ALUMINUM
AD-equivalent neuropathology in the animal EFFICIENTLY AND HAVE HIGHER
model for AD PLASMA AND BRAIN ALUMINUM
LEVELS THAN AGE-MATCHED
Neuropathological examination of sections pro- NON-DEMENTED CONTROLS
cessed with Walton’s stain for aluminum [143]
involved cell counts performed with image analysis Aluminum absorption and plasma aluminum levels
software. These revealed that the superficial entorhinal in AD
cortex in brains from rats with cognitive deteriora-
tion had a significantly larger proportion (60%) of AD patients absorb about 64% more aluminum
pyramidal and stellate cells at stage IV aluminum than age-matched controls from a standardized dietary
aluminum dose [152]. Consequently, AD patients have importance of careful dissection to obtain 10–20 mg
higher plasma/serum aluminum levels than controls samples of gray matter for measurements of aluminum
[153–159]. concentration. Samples larger than 20 mg are likely
Down syndrome is commonly regarded as a model to give erroneous results by diluting the sample with
for accelerated AD. Down syndrome patients have white matter that has lower aluminum content.
even greater aluminum absorption than AD patients Two early studies were unable to confirm higher
from a standardized aluminum dose. Down syn- aluminum values in AD brains than in controls [136,
drome patients absorb approximately 6-fold more 137]. The study by Markesbery et al. [137] utilized tis-
aluminum than age-matched controls from a stan- sue samples ranging from 100 to 250 mg (dry weight).
dardized dietary aluminum dose and 4-fold more Ganrot [171] described methodological flaws in both
from a standardized pharmacological aluminum dose studies. Markesbery’s group [137] subsequently revis-
[160]. ited this issue, using conditions specified in the original
The reason(s) for increased aluminum absorption report (20 mg (dry weight) sample sizes and more
in AD patients and even more so in Down syndrome sensitive instrumentation) and then reported signif-
patients is unknown. The fact that Down syndrome icantly higher aluminum values in biopsy samples
patients absorb much more aluminum than age- of AD hippocampus, inferior parietal lobule, supe-
matched controls from a standardized dose suggests rior and middle temporal gyri than in controls [166].
that the absorption difference could be attributable Brain sections used as non-demented elderly con-
to an intestinal protein encoded by a gene on chro- trols in the McDermott study [136] were re-examined
mosome 21. A␤PP is on chromosome 21 and recent and found to contain abundant NFTs, indicating
evidence indicates that A␤PP-deficient mice demon- that their classification as controls was incorrect
strate impaired intestinal absorption [161]. A␤PP and [172].
A␤ are both expressed in enterocytes [162], the absorp- Aluminum values in gray matter of brains from aged,
tive epithelial cells of the small intestine, so they non-demented humans typically range from about 1 to
are candidates for elevating aluminum absorption. 2.5 ␮g Al/g brain tissue (dry weight) [135, 168]. Alu-
Aluminum has previously been shown to upregu- minum values in gray matter of AD brain are 2- or
late expression of the A␤PP gene [83] and stimulate 3-fold higher, generally around 3 to 7 ␮g Al/g brain
A␤PP mRNA [163] expression in neural cells so tissue [135, 168]. Approximately 20% of samples mea-
aluminum may have the same effects on the A␤PP sured in AD-affected brain regions have aluminum
gene and mRNA expression in intestinal cells. A␤PP values greater than 5.5 ␮g Al/g brain tissue [104, 135].
and/or A␤ have yet to be tested to learn whether Importantly, toxic effects occur in cats at cerebral alu-
either or both are capable of enhancing aluminum minum concentrations between 4 and 6 ␮g Al/g brain
absorption and, if so, the conditions under which tissue [173]. Also, the LD50 (50% lethality dose) for
this occurs. Regardless, higher levels of aluminum brain aluminum in rabbits is already evident at 5.5 ␮g
absorption give rise to higher brain aluminum levels Al/g brain tissue [172]. Aluminum combined with
[146]. food acids, such as aluminum maltol (approved as a
food additive in some countries), are much more toxic
than poorly soluble inorganic aluminum compounds
AD and brain aluminum [172].
A␤PP/tau triple-transgenic (3xTg-AD) [174] mice
Aluminum has been regarded as a candidate cause of are reported to have higher aluminum levels in their
AD since 1973 when it was first shown that gray matter brains than controls, even without aluminum supple-
from brains of AD patients contains more aluminum mentation [175]. It would be interesting to know if
than that of non-demented age-matched controls [135]. other untreated A␤PP-transgenic animal models also
This finding has been confirmed by at least 11 reports have elevated aluminum levels in their brains which
from different researchers working in at least seven dif- would imply more efficient aluminum absorption.
ferent countries using a variety of analytical techniques Down syndrome patients are reported to exhibit
[104, 135, 143, 144, 164–170]. brain aluminum values as high as those in AD brains
Aluminum concentrates in cell bodies localized but at earlier ages [135]. Down syndrome patients gen-
in gray matter. When AD patients were initially erally show AD neuropathology by early middle age
reported to have higher brain aluminum levels than [176] and have a significantly higher rate of AD-type
non-demented controls, the authors emphasized the dementia than age-matched controls [177].
PHYSICAL PROPERTIES OF ALUMINUM Aluminum has also been implicated in other

ACCOUNT FOR ITS NEUROTOXICITY dementias; namely, Balint’s disease from occupational
aluminum exposure [192], amyotrophic lateral scle-
The aluminum ion (abbreviated Al3+ ) has a high rosis/parkinsonism dementia complex of Guam [193,
charge (+3) and a very small ionic radius (0.51 Å) in its 194], and Parkinson’s disease with AD-type demen-
preferred coordination state with six water molecules. tia [195]. Furthermore, aluminum is the acknowledged
Al3+ is smaller than magnesium (Mg2+ ) and slightly cause of cognitive impairment in hundreds of Canadian
smaller than the ferric ion (Fe3+ ) [178]. The size of miners who inhaled McIntyre powder over years in a
Al3+ allows it to effectively compete in cells with the prophylactic attempt to reduce their chance of develop-
essential metals Mg2+ and Fe3+ . ing lung silicosis. The number of miners who became
Mg2+ regulates more than 300 enzymes [179]. cognitively impaired correlated in a time-dependent
Nanomolar amounts of Al3+ successfully compete manner with their length of exposure to McIntyre pow-
with mM amounts of Mg2+ , substituting for Mg2+ der; i.e., from 0.5 to 10 years, 10–20 years or more than
in the active sites of some key regulatory enzymes, 20 years [196]. McIntyre powder consists of poorly
including protein kinase C, and in ATP and GTP soluble aluminum hydroxide and pulverized (metallic)
co-factors. In this way, Al3+ alters, and impairs, the aluminum.
activities of key regulatory proteins and cellular signal-
ing pathways [180, 181 (review)]. Al3+ also substitutes
for Fe3+ in Tf and on IRP2 [182, 183]. EVIDENCE FOR ALUMINUM
Al3+ competes with Mg2+ , Fe3+ , ferrous iron INVOLVEMENT IN AD PATHOGENESIS
(Fe2+ ), and calcium (Ca2+ ) for cell ligands, partic-
ularly phosphates. Al3+ also competes with Ca2+ ions The studies that follow, including some based on
for their sites on membranes and in calcium channels transgenic animal models and others on wild-type ani-
[184–186]. mal models, replicate what appear to be the most
The high charge:size ratio of Al3+ causes it to important neuropathological aspects of AD.
dissociate from ligands very slowly, approximately
108 times more slowly than Ca2+ and 104 times NFT formation
more slowly than Mg2+ [178]. The slow ligand
exchange rates of Al3+ prevent Al3+ from suc- Protein phosphatase 2A activity and mRNA
cessfully participating in enzyme reactions as they levels are diminished in AD, resulting in
require rapid dissociation. Hence, Al3+ disrupts cell hyperphosphorylation of cytoskeletal proteins
metabolism, largely by interfering with the activity of AD brains show depressed activity of protein phos-
key regulatory enzymes and cell signaling processes phatase 2A (PP2A), the main enzyme in mammalian
[180, 182]. brain that removes phosphates from phosphorylated
Another aspect of Al3+ toxicity is its pro-oxidant tau [197], and abnormally low mRNA levels for PP2A
status. Al3+ produces oxidative stress on its own [198]. The low PP2A activity in AD brains results
and synergistically with copper and iron [187–189]. in hyperphosphorylation of cytoskeletal proteins (in
These neurotoxic features of aluminum severely inca- particular, hyperphosphorylated tau and hyperphos-
pacitate neuronal function rather than killing cells phorylated neurofilament protein) in pyramidal cells
outright. and on NFTs [199–206]. Inhibited PP2A activity
resulting from a PP2A mutation in transgenic mice
also correlates inversely with increased levels of hyper-
ALUMINUM INVOLVEMENT IN phosphorylated protein [207].
DEMENTIAS OTHER THAN AD In AD brains, intracellular NFTs at an early stage of
NFT formation are reported to stain more intensely for
High aluminum levels in dialysate water have ele- hyperphosphorylated neurofilament protein and less
vated brain aluminum levels in dialysis patients and for hyperphosphorylated tau. At later stages, the NFTs
caused multiple cases of DES or dialysis dementia stain more intensely for hyperphosphorylated tau and
[111, 190, 191]. Aluminum levels in the brains of less for hyperphosphorylated neurofilament protein
untreated DES patients have exceeded 25 ␮g/g in brain [199]. Some authors [200] have suggested that phos-
tissue with death occurring within months to several phorylated neurofilament protein participates in the
years [191]. formation of human NFTs by serving as a scaffold
for tau deposition since hyperphosphorylated neuro- the increases of hyperphosphorylated tau that occur
filament immunoreactivity appears in patterns (linear in brains of aluminum-exposed renal dialysis patients
skeins, flame-shaped, or intraneuronal globose skeins) [114], Down syndrome patients [209], and AD patients
very similar to those of silver-stained NFTs. Ultra- [115].
structural immunocytochemical studies have shown Immunostaining evidence indicates that relatively
that antigenic determinants in the 200-kDa neuro- small amounts of aluminum are sufficient to inhibit
filament protein, Bodian’s silver-binding sites in the PP2A activity in neurons. Evidence for this is con-
carboxyl-terminal tailpieces of neurofilament subunits tained in serial sections of CA1 pyramidal cells from
and microtubule-associated proteins, especially tau, the brains of wild-type rats chronically exposed to
are all integral components of the filaments that consti- aluminum at human-equivalent levels [212]. Immunos-
tute the NFTs of AD brains [201]. These authors note tained sections show CA1 cells with abundant amounts
that NFT filaments comprise sequences from both tau of hyperphosphorylated tau. CA1 cells in adjacent sec-
and neurofilament proteins in a hyperphosphorylated tions stained for aluminum are mostly at intermediate
form. stages (II–III) of aluminum accumulation [212]. Small
Several studies, for example, that by Ksiezak- amounts of hyperphosphorylated tau are just visible
Reding et al. [208], have reported that immunostaining in some neurons equivalent to those with stage I alu-
for hyperphosphorylated neurofilaments in NFTs is minum accumulation.
artifactual, resulting from antibodies that are cross- Aluminum inhibition of PP2A activity leads to
reactive for both neurofilament and tau proteins. the accumulation of hyperphosphorylated cytoskele-
Vickers et al. [202] analyzed the report by Ksiezak- tal proteins in neurons of humans, rats, rabbits and
Reding and colleagues, noting that those authors in vitro experimental systems [114, 115, 211–219].
had used inappropriately small dilutions to demon- Nascent intracellular tangles, recently induced by alu-
strate cross-reactivity between neurofilament and tau minum in rabbit brain, initially exhibit phosphorylated
proteins. Vickers et al. observed that faint cross- and non-phosphorylated neurofilament protein [215,
reactivity to tau was just visible in an immunoblot 216]. Over the next few days, the rabbit tangles change,
assay with the SM1310 neurofilament antibody if its gradually developing epitopes of hyperphosphorylated
concentration was higher than that normally used. tau, A␤PP, A␤, a1 -antichymotrypsin, and ubiquitin-
However, cross-reactivity for native tau was absent protein conjugates [219]. Hence, NFTs that are rapidly
when all three of their antibodies for hyperphos- induced in certain animal brains by intracerebral alu-
phorylated neurofilaments (SM1310, SM 132, and minum injection evolve to resemble human NFTs [199]
M14) were used at appropriate dilutions. Also, Vickers more than commonly acknowledged.
et al. used a double-labeled immunostaining tech- A consequence of tau hyperphosphorylation
nique that revealed NFTs are immunoreactive both for induced by aluminum in neurons is that the excess
hyperphosphorylated tau and for hyperphosphorylated phosphate interferes with tau’s ability to bind to
neurofilament protein [202]. microtubules. This increases the level of soluble
Down syndrome patients likewise exhibit a dramatic hyperphosphorylated tau in the cytoplasm [220]. The
decrease in PP2A levels which correlates inversely cell continues to produce more normal tau, which
with elevated levels of hyperphosphorylated tau soon becomes hyperphosphorylated, contributing to
[209] and hyperphosphorylated neurofilament protein the increasing accumulation of hyperphosphorylated
[210]. tau. Aluminum causes hyperphosphorylated tau to
aggregate into granules of increasing size while
Aluminum inhibits PP2A activity in vitro and in sparing normal tau [218].
vivo, resulting in hyperphosphorylation of Around this stage, caspase truncates hyperphospho-
cytoskeletal proteins rylated tau [221] in AD cells. Aluminum activates
Aluminum inhibition of PP2A activity results in the caspase 3 [222, 223], the same type of caspase involved
in vitro formation of hyperphosphorylated tau, the key in tau truncation [221, 224]. Since aluminum activates
protein of mature NFTs [211]. Aluminum-inhibited caspase 3, inhibits PP2A activity [211, 212], thereby
PP2A activity also results in the accumulation of hyper- promoting the accumulation of hyperphosphorylated
phosphorylated tau in brains of wild-type rats exposed tau, and is present in AD cells where NFTs form,
to human-equivalent levels of aluminum for most of aluminum may also participate in caspase-induced
their adult life [146, 212], suggesting that aluminum- truncation of hyperphosphorylated core tau in these
induced inhibition of PP2A activity may also explain cells.
Aluminum induces the formation of NFTs from

hyperphosphorylated tau in brains of humans,
particularly those with AD
I. At the pre-tangle stage, aluminum avidly binds to
phosphates on the hyperphosphorylated tau, caus-
ing this form of tau to aggregate, precipitate, and
form granules of increasing size in AD hippocam-
pal and cortical neurons [218, 225] (Fig. 7A, B).
II. By the late pre-tangle stage, the aggregated gran-
ules have coalesced to form a homogeneous,
confluent cytoplasmic mass that stains for both
aluminum and hyperphosphorylated tau [225]
(Fig. 7C, D)
III. At an early tangle stage, nascent filaments
can be seen within the otherwise homoge-
neous aluminum/hyperphosphorylated tau com-
plex (Fig. 7E, F).
IV. As more filaments precipitate within the complex,
they grow and form NFTs that continue to stain
for both aluminum and hyperphosphorylated tau
[225] (Fig. 7G, I).
These NFTs are the markers used by neuropatholo-

gists to identify AD-affected brain regions involved in
AD progression. The aluminum component of NFTs
has been confirmed by several different types of instru-
ments [169, 170] as well as staining techniques. Some
iron is also found in NFTs [67], suggesting that NFTs
are a cell-protective response, involving sequestration
of potentially-toxic free metal ions. Aluminum binding
to growing NFTs in the neuronal cytoplasm may be a
cellular mechanism for slowing aluminum uptake into
the nucleus where aluminum avidly binds to nucleic
acids and chromatin [225].
Only a portion of the large CA1 neurons in AD-
affected human brains, and aluminum-exposed rabbits,
form NFTs [225, 226], indicating that the conditions
for NFT formation are rather specific. For species
reasons, NFTs are absent from virtually all aged rat
neurons, even in those that contain abundant amounts
of aluminum and hyperphosphorylated tau [212].
Instead, aged rat hippocampal and cortical neurons Fig. 7. NFT formation in AD-affected hippocampus of human brain.
A, B) Pre-tangle pyramidal cells stained for hyperphosphorylated tau
show various stages of nuclear aluminum accumula- show (A) small and (B) larger (fused) granules (arrows). C, D) Cyto-
tion as do many hippocampal and cortical neurons of plasmic pools form from granule fusion and stain for (C) aluminum
aged humans (Fig. 8) and aluminum-exposed rabbits and (D) hyperphosphorylated tau. Arrows mark the thinner margins
[140, 143, 226]. of the cytoplasmic pools that consist of an Al/hyperphosphorylated
tau complex. E, F) Thin filamentous structures just visible in the
It would be rare for any single animal model to cytoplasmic pools, stained for (E) aluminum and (F) hyperphospho-
replicate all aspects of a complex disease like AD. rylated tau, represent nascent filaments that develop into NFTs. G,
For example, A␤PP- and tau-transgenic mice are use- H) Mature NFTs continue to stain for (G) aluminum and (H) hyper-
phosphorylated tau. I) A large NFT stained to show aluminum (pale
ful models for understanding amyloidogenesis and
purple filaments) has consumed the cytoplasmic pool so individual
tauopathies, respectively, in that they focus on replicat- filaments are more clearly seen (arrow). MB = 7 ␮m for A–H; 3 ␮m
ing a specific AD hallmark rather than AD progression. for I. Reproduced from [225] with permission from IOS Press.
Fig. 8. Stages of aluminum accumulation in rat and human hippocampal pyramidal neurons. Stage 1: All sections of hippocampal pyramidal
neurons from older human brain, that contain a visible nucleolus, exhibit at least stage I aluminum accumulation (staining only the nucleolus).
Stage I neurons have a normal shape. Stages II and III: The nucleoplasm progressively deepens in hue, appearing pink at stage II and purple at
stage III. The nucleus and overall cell shape show subtle shrinkage and dendritic changes. Stage IV: Aluminum in the nucleus appears bright
magenta. The cell is obviously shrunken and its neurites appear tortuous and retracted. At stage V, aluminum is distributed throughout the
nucleus and cytoplasm. These cells are deformed but appear to be viable, exhibiting neither necrosis nor apoptosis. Magnification bar = 5 ␮m.
Reproduced from [143] with permission from Elsevier.
The aging aluminum-based rat model is valu- [228] and this paper has already addressed aluminum
able in that it replicates key aspects of AD clinical participation in human NFTs [225]. The aluminum-
and neuropathological progression. That is, aluminum induced rat model shows neuropathology that can
specifically produces severe neuropathological dam- account for dendritic/axonal dieback, synapse loss, and
age to the cells of origin for the perforant pathway cortical atrophy, including that which appears on MRI
and hippocampal pyramidal cells in the rats that devel- scans analyzed for AD progression [143].
oped cognitive deterioration. Damage to these cells
and brain regions in humans with AD isolates the hip- High stage aluminum accumulation in cells and
pocampus from the neocortex and impairs memory its consequences
function [25, 29, 227]. High stage aluminum accumulation refers to intense
Hippocampal isolation can logically explain the staining for aluminum in the cell nucleus (Stage IV)
cognitive deterioration that occurred in a substantial or throughout the nucleus and cytoplasm (stage V)
proportion of the rats that chronically consumed alu- (Fig. 8) [143]. The available evidence indicates that
minum at the high end of the human total dietary aged corticocortical cells with high stage aluminum
aluminum range. The rats with cognitive deterioration accumulation, whether in the form of NFTs or nuclear
had severely impaired memory function and they dis- aluminum, are drastically different cells from their
played other AD-type behaviors and neurological signs healthier counterparts that exhibit low-stage aluminum
[41, 143, 146]. Furthermore, the sequence of damage in accumulation, both with respect to structure and
their brains parallels that which occurs with AD pro- function.
gression. The superficial entorhinal cortex was most An electrophysiological consequence of Al-induced
damaged, followed by other limbic regions and neocor- NFT formation in neurons that normally discharge
tical association areas—in that order—and, to a much at high spontaneous frequencies is that they become
lesser extent, motor and primary sensory areas. The electrically inactive [173]. Human neurons with NFTs
animal model replicates cell damage in the sequence have low cytochrome oxidase activity [229]. Low
of brain regions involved in AD progression. concentrations of aluminum inhibit mitochondrial
As for AD hallmarks, the aging aluminum-based rat cytochrome c oxidase activity. Low cytochrome c oxi-
model for AD shows incipient changes in A␤ and NFT dase activity indicates a low metabolic rate, suggesting
formation. These changes seldom if ever develop into these cells are no longer capable of neuronal function
mature plaques and tangles, clearly a result of species [230, 231]. Dysfunctional human cells with NFTs are
differences. However, aluminum participation in the able to survive in this diminished condition in brain
formation of A␤ and A␤ plaques is described below tissue for decades [232]. Dysfunctional cells with high
Fig. 9. High stage aluminum accumulation correlates with microtubule depletion in rat cortical and hippocampal cells. A) Stage IV pyramidal
cells stain magenta for nuclear aluminum within a hippocampal CA1 lesion of an aged rat with cognitive deterioration. Pyramidal cells with a
more normal appearance (arrow) are seen at the margins of the lesion. B) An adjacent section immunostained for acetylated-␣-tubulin shows
that cells within the same lesion fail to immunostain for microtubules whereas microtubules can be clearly seen in the more normal cells along
the margins of the lesion (arrow). Magnification bars = 10 ␮m. Reproduced from [143] with permission from Elsevier.
stage nuclear aluminum accumulation likewise persist

in animal brains for a prolonged time [143].
Human and/or rat pyramidal cells that exhibit NFTs
or high stage nuclear aluminum accumulation consis-
tently show microtubule depletion (Fig. 9) [143, 233,
234]. Aluminum-induced microtubule depletion leads
to: shrinkage and change in cell shape [140, 143];
impaired axonal transport [163, 235]; dendritic/axonal
dieback [143, 236, 237]; and significant loss of synapse
density [143, 238, 239]. Dieback results in shrinkage of Fig. 10. Camera lucida drawings of AD Golgi-stained pyramidal
cells illustrating the process of dendritic/axonal dieback (left to
the cell’s dendritic tree (Fig. 10) that normally accounts
right). Redrawn from [241] with permission from Elsevier.
for 95% of cell volume [240]. Widespread dieback
leads to significant cortical atrophy [241] that becomes
increasingly extensive as AD/chronic aluminum neu-
rotoxicity spreads more widely, in a chain reaction that granule, 0.5 to 1.5 ␮ across. GVD occurs in most
involves increasing numbers of brain regions. AD patients, affecting 9–66% of hippocampal pyra-
midal cells [243]. Granules in the vacuoles of
human hippocampal neurons stain for aluminum [140]
Aluminum involvement in granulovacuolar and exhibit an aluminum peak when examined by
degeneration (GVD) energy dispersive X-ray microanalysis spectroscopy
[244]. GVD granules also stain for altered proteins,
Simchowicz, one of Alzheimer’s students, reported mainly caspase-cleaved A␤PP and hyperphosphory-
that hippocampal cells of AD tissue show GVD lated tau. GVD formation is experimentally induced
[242]. This pathological feature consists of clus- in hippocampal cells of rats chronically exposed to
ters of intracytoplasmic vacuoles, measuring up to dietary aluminum at human-equivalent levels [146,
5 ␮ in diameter, that each contain a single dense 212] or by repeated intraperitoneal injections of
Fig. 11. The A␤ portion of the rat A␤PP sequence. The protein sequence is shown in the standard one-letter code below the nucleotide sequence.
The rat equivalent of the human A␤ polypeptide is located between amino acid residues 598–640 and is shown in gray. Also bold/highlighted
are the three positions (601, 606, and 609) where the rat and human sequences differ. The human amino acid residues are indicated below the
rat amino acid residues at these positions. The highlighted residues indicate the presumed transmembrane region. Reproduced from [256] with
permission from the Nature Group.
aluminum [245]. Aluminum appears to be the only providing that phosphorylation by protein kinase C
agent reported to experimentally induce GVD. GVD (PKC) has occurred [249, 250]. This cleavage pre-
also occurs together with NFTs in amyotrophic lat- cludes the formation of A␤. However, aluminum, at
eral sclerosis/parkinsonism dementia of Guam [193, a concentration of only 2 × 10−8 mol/L, inhibits 90%
246], another neurodegenerative disease that involves of PKC activity [251]. Thus, a small amount of intra-
chronic aluminum neurotoxicity. neuronal aluminum has the potential to redirect A␤PP
cleavage from its non-amyloidogenic pathway (form-
Aluminum involvement in Aβ metabolism ing sA␤PP␣) to its amyloidogenic pathway (forming
A␤). This can explain why exposure of cultured rat
A␤ deposits develop under multiple conditions: as neurons to 10–50 ␮M aluminum for three weeks results
part of the degenerative process in regions of brain in a pronounced increase in soluble A␤ in addition to
where terminals are deteriorating [29]; in association tau accumulation and neurite degeneration [252].
with some blood vessels [247]; and in response to
oxidative stress [228]. Aluminum is present in large
corticocortical neurons of AD patients [140] and can Aluminum interactions with Aβ
interact with A␤ at various stages of its formation in Aluminum forms complexes with A␤ in vitro and
experiments. Hence, aluminum may interact with A␤ stabilizes A␤ oligomers [253]. Aluminum also con-
in the same way in elderly human brains. verts soluble human A␤42 with random-coil structure
to a fibrillar precipitate with ␤-pleated structure [254,
255] and induces human A␤ fibrils to aggregate [248].
Aluminum upregulation expression of the AβPP
A␤ fibrillization is a species-specific effect that occurs
gene, its mRNA and protein
in animal species that have the same amino acid
Aluminum upregulates gene expression for A␤PP
sequence for A␤ as in humans. The A␤ sequence of rats
and other stress-response proteins in human neural
and mice differs from the A␤ sequence for humans by
cells [83]. A␤PP mRNA and A␤PP protein expression
three amino acids (Fig. 11) [256]. This species-specific
are increased in the brains of rats chronically exposed
difference is apparently sufficient to block soluble A␤
to aluminum levels equivalent to those consumed by
from fibrillizing and forming A␤ plaques in mouse and
humans [163]. Aluminum induces axonopathy in hip-
rat brains [235, 256].
pocampal and cortical regions of rat and rabbit brains in
which A␤PP-immunostained axons show constrictions
and varicosities, indicating impaired axonal transport Aluminum accelerates and enhances Aβ plaque
[163, 235]. formation in a transgenic mouse model for
amyloidogenesis
Aluminum alters AβPP cleavage to form Aβ Mice have been genetically-engineered to overex-
instead of sAβPPα press human A␤PP and to form human A␤. This allows
A␤PP is cleaved by ␣-secretase at a site that results A␤ plaques to form in brains of relatively young trans-
in the formation of sA␤PP␣ which is neurotrophic, genic mice. Human A␤PP-transgenic mice, fed a diet
supplemented by aluminum for one year, exhibited Aluminum induces the formation of a presenilin-2
significantly higher levels of plasma A␤ as well as sol- variant that increases Aβ42 and disrupts signaling
uble and fibrillar forms of A␤ in their brains [228]. in the hippocampal CA1 field of sporadic AD
A␤ plaque formation occurred earlier and in appre- brains
ciably larger amounts in brains of aluminum-exposed
transgenic mice than in brains of a transgenic cohort The AD research community has long focused on a
without aluminum supplementation. Thus, aluminum search for mutations in three main candidate genes:
stimulates the formation of A␤, both in oligomeric and those for A␤PP, presenilin-1, and presenilin-2. The
plaque forms, in the brains of A␤PP-transgenic mice presumed defect would enhance A␤42 levels in the
that express human A␤PP and the human A␤ peptide. brain and distinguish sporadic AD cases from non-
An implication of this study is that the presence of demented controls. A presenilin-2 gene product that
aluminum in AD brains could likewise stimulate the satisfies these requirements was identified by a group
formation of human A␤. of researchers around the turn of the 21st century [262,
Congophilic amyloid angiopathy (CAA), a common 263].
accompaniment to AD, involves A␤ deposits in cor- This gene product comes from an inducible,
tical blood vessels. A␤ also deposits in the walls of alternatively-spliced variant of the presenilin-2 gene
cortical vessels of mice that have drunk water con- designated PS2V rather than from a mutant gene. The
taining alum for at least one year, serving as a model exon 5 sequence is omitted during transcription of the
for CAA in humans [257]. The first neuropatholog- PS2 gene [262]. The shortened mRNA that encodes
ical examination of a person overexposed to alum PS2V is truncated at its N-terminus. PS2V is reported
from the Camelford water pollution incident, who died to be present in the brains of all sporadic AD cases
of an unspecified neurological condition, was deter- examined by the authors and absent from 90% of brains
mined to have a rare form of sporadic early-onset CAA from non-demented controls [263]. PS2V increases
in leptomeningeal and cerebral cortical vessels. High A␤42 production by affecting A␤PP cleavage [262].
concentrations of aluminum were found in severely- PS2V also interferes with A␤PP maturation and dis-
affected cortical regions of this patient’s brain [258]. rupts the signaling pathway for the unfolded protein
response, particularly in the hippocampal CA1 field
Aluminum in Aβ plaque cores where PS2V is associated with dendritic dieback [263].
A recent study by Yumoto et al. [259] using trans- PS2V expression is inducible in cultured cells by
mission electron microscopy with an ultrasensitive hypoxia and can be blocked by antioxidants. This sug-
spectrometric technique reported that aluminum is gested to the authors that the PS2V isoform could
detectable in the cores of A␤ plaques. The present be induced by a metal that causes oxidative damage.
author used histological processes to find aluminum Neuroblastoma cells were exposed to copper chloride,
in AD neurons and NFTs. It is clear that ultra- copper sulfate, zinc chloride, ferrous chloride, ferric
sensitive highly sophisticated precision techniques chloride, aluminum chloride, and aluminum maltolate
such as that of Yumoto need to be brought to bear to learn whether they might be involved in PS2V for-
to determine the presence of minute amounts of mation. Aluminum (both as the chloride and maltolate
aluminum in plaques and the significance of that salts) is the only metal that consistently induces the
presence. PS2V isoform and does so at a low (25 ␮M) aluminum
A different group, using a prototype nuclear micro- concentration (Al) [264].
scope, reported 15 years earlier that aluminum is
absent from A␤ plaques [260]. A paper published by
the latter authors on the same subject in the follow- Inflammation in an aluminum-based animal model
ing year [261] contained discrepancies; for example, for AD
reporting their nuclear microscope had a resolu-
tion of 50 ppm compared to the 15 ppm resolution Aluminum salts contained in drinking water, at
reported in the previous year. The nuclear micro- amounts to which humans are routinely exposed, were
scope has yet to be validated for its ability to detect found to increase cerebral levels of glial activation and
aluminum in histological tissue sections. Regardless, inflammatory cytokines as well as increasing the level
Kawahara et al. report that aluminum concentrations of A␤PP within brains of mice [265]. AD is known
lower than 15 ppm are sufficient to aggregate A␤ to be a mild inflammatory condition associated with
[248]. elevating basal levels of inflammation markers.
ALUMINUM AND THE FEATURES

PROPOSED TO INCREASE CELL
SUSCEPTIBILITY TO NFT FORMATION
Aluminum, large cell size, and complexity
Histological stains and SIMS analyses for aluminum

in brain tissue from AD cases and animal models for
AD reveal that aluminum preferentially accumulates
in large corticocortical cells of the entorhinal cortex,
hippocampal CA1 field, subiculum, amygdala, and
neocortical pyramidal cells with long corticocortical
projections in layers III and V [41, 140, 143, 146,
150, 151].
Aluminum and high levels of metabolic activity,

transferrin receptors, and iron uptake
Aluminum metabolism is closely intertwined with

iron metabolism. The close physical resemblance
between Al3+ and Fe3+ ions (charge density, ionic
size, and favored coordination number) allows Al3+
to mimic Fe3+ in plasma and cells. Cellular proteins
involved in iron transport and iron uptake into cells are
somewhat promiscuous in that they fail to discriminate Fig. 12. Stabilized translation of Tf receptor mRNA under patholog-
between Fe3+ and Al3+ . This may explain why approx- ical circumstances. A) Fe3+ ion oxidation of IRP2 does not occur in
imately 90% of plasma aluminum binds to unoccupied AD-affected neurons despite high iron levels. This results in stabi-
lized translation of Tf receptor mRNA and continuation of iron and
iron-binding sites on Tf, circulating as Tf-aluminum,
aluminum uptake. B) Aluminum ions bind to Fe3+ ion-binding sites
with the remainder binding to albumin and low molec- on IRP2s of aluminum-exposed neurons, preventing Fe3+ ions from
ular weight species such as citrate [266, 267]. oxidizing the IRP2s and signaling for IRP2 destruction. This results
Aluminum utilizes endothelial Tf receptors, evolved in the stabilized translation of Tf receptor mRNA and continuation
of iron and aluminum uptake. In AD and aluminum exposed cells,
for iron uptake, to cross the blood-brain barrier [49, IRP1 behaves in its normal manner (see Fig. 3C and D).
268] and neuronal Tf receptors for entering large
complex neurons [53]. Aluminum has at least three
modes for uptake into neurons: (1) trans-synaptic
transport between interconnected neurons; (2) Tf- is much higher than for other metals, including iron.
mediated endocytosis; and (3) mechanisms involved Aluminum-induced stabilization of IRP2 prevents
in Tf-independent iron uptake (Tf-IU) [269]. Alu- IRP2 oxidation and degradation in the ubiquitin-
minum preferentially deposits in large cells with high proteosomal pathway (Fig. 12B). This allows IRP2
metabolic demands, high densities of Tf receptors on to stay bound to the mRNA for Tf receptors which
their plasma membrane surface, and high iron uptake in turn allows Tf receptor production to continue and
to meet those metabolic demands [50]. ferritin synthesis to remain suppressed as though the
affected cells were permanently iron-deficient [62, 63].
Aluminum accumulation in neurons leads to iron Aluminum-induced stabilization of IRP2 is consistent
disregulation with the IRP2 stabilization that occurs in AD [65] and
Iron homeostasis becomes disregulated in AD brains may account for this phenomenon. In contrast, IRP1 is
as a result of IRP2 stabilization in large neurons unaltered by aluminum exposure or AD [65, 183].
(Fig. 12A). Micromolar amounts of aluminum (4 In vitro and in vivo evidence both attest to alu-
to 20 ␮M Al) successfully compete with Fe3+ for minum’s effects on iron metabolism. Intraneuronal
iron-binding sites on IRP2. This stabilizes IRP2 and aluminum accumulation enhances iron uptake and
prevents its degradation. According to Yamanaka et al. expression of hyperphosphorylated tau in neuroblas-
[183], the affinity of Al3+ for these iron-binding sites toma cells [270]. A kinetic radiolabelled Tf-Fe binding
assay showed the number of surface Tf receptors per more energy to compensate for its axonal wastage.
cell was higher in cells cultured in the presence of The entry of aluminum and excess iron into the
aluminum than in those without aluminum exposure poorly-myelinated neurons of the entorhinal cortex,
[271]. This increase in Tf receptor numbers probably hippocampus, and other limbic regions could render
reflects aluminum-induced stabilization of IRP2. these neurons susceptible to significant peroxidative
Rats given a series of thrice weekly intraperi- stress and its oxidative consequences—oxidized fats
toneal injections of aluminum gluconate over 8 weeks and proteins, leading to the formation of reactive alde-
(“aluminum-loading”) showed dramatically increased hydes and carbonyls, cross-linking of proteins, nucleic
brain aluminum levels, ranging between 14 to 25 ␮g/g acids, and proteins with nucleic acids [277, 278].
(wet weight) in AD-vulnerable brain regions. The high
aluminum levels were paralleled by increased iron lev-
Aluminum and vascularization
els in the same regions, including 4- to 9-fold iron
increases in the temporal, frontal, and parietal cor- Tf receptor densities on capillary endothelium are
tices and a 4-fold iron increase in the hippocampus 6 to 10 times greater than those on neurons [279].
of aluminum-exposed rats compared to iron levels in The vascular plexus that surrounds stellate and pyra-
untreated controls [272]. These results suggest that the midal cell islands of the superficial entorhinal cortex
subtle rise in brain iron stores that occurs for some time invests the islands with dense capillary networks [1].
with increasing age [103, 273] may be secondary to the In principle, larger amounts of aluminum should be
increase that occurs in brain aluminum levels over time removable from the plasma by these dense capillary
[135–138]. networks and transferred into the extracellular matrix
surrounding the cells of the superficial entorhinal cor-
Aluminum, iron, and oxidative stress tex than in regions where capillaries are less abundant.
Aluminum produces oxidative damage on its own Increased amounts of extracellular aluminum would
and synergistically with iron. Intracellular aluminum then be available to plasma membrane Tf receptors
forms an aluminum-superoxide complex that pro- for uptake into neurons. This may be a reason why
duces oxidative damage [187, 274] and aluminum also aluminum preferentially accumulates in the entorhinal
enhances iron-initiated oxidative damage [187, 275]. cortex early in AD and why these cells are damaged to
Brain cells have a limited ability to react to oxidative a greater extent than cells of other limbic regions.
stress, particularly in view of their relatively low levels
of glutathione and glutathione peroxidase.
Aluminum-loading for eight weeks significantly ALUMINUM AND AD PROGRESSION
increases levels of lipid peroxidase while decreasing
superoxide dismutase and glutathione peroxidase lev- Aluminum can spread from one brain region
els even further. Iron elevation on its own is unable to another via interconnected corticocortical cells.
to produce consistent change in any of the cytopro- Gelfoam® impregnated with 15% aluminum lac-
tective enzymes (superoxide dismutase, glutathione tate or 5% aluminum chloride was implanted into
reductase, glutathione peroxidase, and catalase) [272]. the nasal cavity of rabbits [280]. One month later,
Aluminum stabilizes intracellular ferrous iron by high aluminum levels and neuropathological changes
preventing its oxidation to ferric iron [275, 276]. Fer- were observed in the olfactory bulb, pyriform cortex,
rous iron reacts with hydrogen peroxide, driving the hippocampus, and cerebral cortex. This study demon-
highly toxic Fenton reaction that generates reactive strates that soluble forms of aluminum can enter the
hydroxyl radicals that increase oxidative stress. Alu- processes of olfactory neurons in the nasal mucosa and
minum stabilization of the ferrous iron is an important move across pyramidal cell synapses in both retrograde
mechanism for enhancing iron-induced free radical and anterograde directions [26].
production and oxidative stress [276]. Aluminum spread to different brain regions via
interconnected corticocortical cells, together with
Aluminum and myelination aluminum-inhibited PP2A activity which results in
hyperphosphorylated tau, can logically account for the
Myelin sheaths provide insulation for nerve fibers, “spread of hyperphosphorylated tau” reported to occur
allowing impulses to transmit quickly and efficiently between interconnected corticocortical cells [35–37].
along the length of the axon. A poorly-myelinated It would be prudent to stain for aluminum, in investi-
neuron with a leaky axon needs to generate vastly gations into the spread of hyperphosphorylated tau, to
ensure aluminum absence before assuming that hyper- Hyperphosphorylated tau is unable to bind to
phosphorylated tau is acting on its own. Aluminum microtubules so microtubule assembly cannot proceed
spread between interconnected cells could also con- and existing microtubules break down. Consequently,
tribute towards the spread of NFTs and microtubule cells that develop hyperphosphorylated tau, including
depletion in AD-affected brain regions [14, 143]. those in which NFTs form, become microtubule-
depleted [233, 234]. Microtubule depletion results in
dendritic/axonal dieback, de-efferentation, and deaf-
SUGGESTED SCENARIO FOR ALUMINUM ferentation of the superficial entorhinal cortical cells.
INVOLVEMENT IN AD PROGRESSION Dieback of cell processes, and neuron death in regions
where abundant NFTs form, appear in MRI scans as
Aluminum ingestion and absorption is an ongoing gray matter atrophy.
process in contemporary society as is aluminum uptake Meanwhile, aluminum aggregates the soluble
into the brain [133]. Dietary aluminum can thus fuel hyperphosphorylated tau that occurs in some cells
the spread of aluminum over time into an increasing [218], forming granular precipitates which fuse to
number of brain regions. Minute amounts of aluminum form larger granules. Eventually, the large granules
progressively enter neurons via Tf receptors, under also fuse to form cytoplasmic pools of the alu-
normal control of the IRPs, throughout the long pro- minum/hyperphosphorylated tau complex out of which
dromal phase. filaments and NFTs precipitate. The NFTs mature,
Intraneuronal aluminum spreads between stellate slowly growing as they acquire more aluminum and
and pyramidal corticocortical cells of the superfi- more hyperphosphorylated tau (Fig. 8G–I). Very large
cial entorhinal cortex, within islands and between NFTs eventually denucleate their host cells which con-
islands, and from these entorhinal cortical cells into sequently die and leave the NFTs behind [140].
connecting corticocortical cells of the hippocampal Such destruction of the entorhinal cortical cells of
CA1/subiculum zone, olfactory bulb, neocortex, origin for the perforant pathway, which bi-directionally
amygdala, and other brain regions with which the interconnects the hippocampal formation with the
entorhinal cortex communicates. The amount of rest of the cortex, results in isolation of the hip-
aluminum provided by projections from the entorhinal pocampus, ultimately leading to the memory defects
cortex to interconnected corticocortical cells in and confusion that herald the onset of overt AD
other brain regions could be proportional to their [25, 29, 227].
connections. The disease continues to spread to corticocorti-
More than midway through the prodromal period, cal cells in other brain regions, exhibiting the same
aluminum accumulation in entorhinal cortical stellate types of pathology and functional loss as in regions
and pyramidal cells begins to reach neurotoxic lev- affected at an earlier stage. In time, the transformation
els. At this point, intracellular aluminum levels are of neurons into dysfunctional cells becomes extensive
sufficiently high to compete with essential metals, to in some AD-affected regions. Microtubule-depleted
inhibit PP2A activity, and disrupt iron metabolism. corticocortical cells in tissues with slower aluminum
Cellular IRP2 levels become stabilized and their iron accumulation and more slowly-growing NFTs, can
metabolism is disregulated, showing an increased survive for decades in the dysfunctional state [232].
density of Tf receptors on their surface. The dis- Microtubule-depleted cells are unable to contribute to
regulated entorhinal cortical cells continue to import neural function. These cells are unable to sustain their
aluminum and iron from the neuropil, exporting these high cytochrome oxidase activity [44, 229]. Most of
metals directly into connecting cells without obvious their chromatin is in the heterochromatin form (com-
restraints on this process. pacted DNA with reduced transcription) [278]. As each
Neurons require tau protein for microtubule assem- of the regions involved in AD progression becomes
bly and microtubule stabilization. The activity of affected by these changes, their specialized functions
tau protein depends on alternating phosphorylation diminish to the extent that their large neurons are
and dephosphorylation reactions. Kinases continue damaged.
to phosphorylate tau but PP2A activity is now too The scenario presented here is consistent with the
low in these cells to remove phosphate groups. This atrophy of gray matter that occurs over time with AD
situation leads to cytoplasmic accumulation of hyper- progression as described by the Braak hierarchical
phosphorylated cytoskeletal proteins; in particular, model, MRI/VBM clinical trials and other neuropatho-
hyperphosphorylated tau [281–283]. logical studies.
CONCLUSIONS and, in principle, spread more widely as AD progresses

throughout the brain.
This paper describes studies that have endeavored
to define the sequence of interconnected brain regions ACKNOWLEDGMENTS
affected in AD, either by MRI/VBM or neuropatho-
logical techniques. There is a consensus among these The author is grateful to Don Bryson-Taylor for his
investigators that AD spreads in a stepwise manner editorial assistance and to Dirce Everett for her scien-
along well-defined corticocortical connections from tific illustrations.
the entorhinal cortex to other limbic regions in the The author’s disclosure is available online (http://
medial temporal lobe, followed by the temporopari- www.j-alz.com/disclosures/view.php?id=1637).
etal association cortices, and then frontal and occipital
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Journal of Alzheimer’s Disease 35 (2013) 7–43 7

Aluminum’s Involvement in the Progression

Accepted 10 January 2013

INTRODUCTION Epidemiological studies for AD, based on identical

SEQUENCING THE BRAIN REGIONS Cell–cell connectivity and AD neuropathological

gyrus. Tau pathology in limbic regions precedes A␤ A consensus on AD neuropathological progression

inhibitory interneurons seldom, if ever, contain NFTs

High energy demands, densities of transferrin (Tf)

A second possibility for increased cell vulnerabil-

distribution to those with neurons that have with high

Iron regulatory proteins (IRPs)

susceptible to oxidative damage. Consequently, those

Redundant vascularization may increase cell

The final feature considered here that may increase a

accumulation compared to 23% at this stage in low

PHYSICAL PROPERTIES OF ALUMINUM Aluminum has also been implicated in other

Aluminum induces the formation of NFTs from

These NFTs are the markers used by neuropatholo-

stage nuclear aluminum accumulation likewise persist

ALUMINUM AND THE FEATURES

Aluminum, large cell size, and complexity

Histological stains and SIMS analyses for aluminum

Aluminum and high levels of metabolic activity,

Aluminum metabolism is closely intertwined with

CONCLUSIONS and, in principle, spread more widely as AD progresses

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