Tau Protein Hyperphosphorylation and Aggregation in Alzheimer’s Disease and Other Tauopathies, and Possible Neuroprotective Strategies

Abstract

Abnormal deposition of misprocessed and aggregated proteins is a common final pathway of most neurodegenerative diseases, including Alzheimer’s disease (AD). AD is characterized by the extraneuronal deposition of the amyloid β (Aβ) protein in the form of plaques and the intraneuronal aggregation of the microtubule-associated protein tau in the form of filaments. Based on the biochemically diverse range of pathological tau proteins, a number of approaches have been proposed to develop new potential therapeutics. Here we discuss some of the most promising ones: inhibition of tau phosphorylation, proteolysis and aggregation, promotion of intra- and extracellular tau clearance, and stabilization of microtubules. We also emphasize the need to achieve a full understanding of the biological roles and post-translational modifications of normal tau, as well as the molecular events responsible for selective neuronal vulnerability to tau pathology and its propagation. It is concluded that answering key questions on the relationship between Aβ and tau pathology should lead to a better understanding of the nature of secondary tauopathies, especially AD, and open new therapeutic targets and strategies.

Full text

biomolecules
Review
Tau Protein Hyperphosphorylation and Aggregation
in Alzheimer’s Disease and Other Tauopathies, and
Possible Neuroprotective Strategies
Goran Šimi´c 1,*, Mirjana Babi´c Leko 1, Selina Wray 2, Charles Harrington 3, Ivana Delalle 4,
Nataša Jovanov-Miloševi´c 1, Danira Bažadona 5, Luc Buée 6, Rohan de Silva 2,
Giuseppe Di Giovanni 7,8, Claude Wischik 3and Patrick R. Hof 9,10
Received: 2 November 2015; Accepted: 1 December 2015; Published: 6 January 2016
Academic Editor: Jürg Bähler
1
Department of Neuroscience, Croatian Institute for Brain Research, University of Zagreb School of Medicine,
Zagreb 10000, Croatia; mbabic@hiim.hr (M.B.L.); njovanov@hiim.hr (N.J.-M.)
2Reta Lila Weston Institute and Department of Molecular Neuroscience, UCL Institute of Neurology,
London WC1N 3BG, UK; selina.wray@ucl.ac.uk (S.W.); r.desilva@ucl.ac.uk (R.S.)
3School of Medicine and Dentistry, University of Aberdeen, Aberdeen AB25 2ZD, UK;
c.harrington@abdn.ac.uk (C.H.); cmw@taurx.com (C.W.)
4Department of Pathology and Laboratory Medicine, Boston University School of Medicine,
Boston 02118, MA, USA; idelalle@bu.edu
5Department of Neurology, University Hospital Center Zagreb, Zagreb 10000, Croatia; b.danira@gmail.com
6
Laboratory Alzheimer & Tauopathies, Université Lille and INSERM U1172, Jean-Pierre Aubert Research Centre,
Lille 59045, France; luc.buee@inserm.fr
7Department of Physiology and Biochemistry, Faculty of Medicine and Surgery, University of Malta, Msida,
MSD 2080, Malta; giuseppe.digiovanni@um.edu.mt
8School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK
9Fishberg Department of Neuroscience, Ronald M. Loeb Center for Alzheimer’s Disease,
Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA; patrick.hof@mssm.edu
10 Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
*Correspondence: gsimic@hiim.hr; Tel.: +385-1-459-6807; Fax: +385-1-459-6942
Abstract:
Abnormal deposition of misprocessed and aggregated proteins is a common final pathway
of most neurodegenerative diseases, including Alzheimer’s disease (AD). AD is characterized
by the extraneuronal deposition of the amyloid
β
(A
β
) protein in the form of plaques and the
intraneuronal aggregation of the microtubule-associated protein tau in the form of filaments. Based
on the biochemically diverse range of pathological tau proteins, a number of approaches have been
proposed to develop new potential therapeutics. Here we discuss some of the most promising ones:
inhibition of tau phosphorylation, proteolysis and aggregation, promotion of intra- and extracellular
tau clearance, and stabilization of microtubules. We also emphasize the need to achieve a full
understanding of the biological roles and post-translational modifications of normal tau, as well
as the molecular events responsible for selective neuronal vulnerability to tau pathology and its
propagation. It is concluded that answering key questions on the relationship between A
β
and tau
pathology should lead to a better understanding of the nature of secondary tauopathies, especially
AD, and open new therapeutic targets and strategies.
Keywords:
Alzheimer’s disease; amyloid
β
; neurofibrillary degeneration; microtubules;
neuropathology; phosphorylation; protein aggregation; protein oligomerization; tauopathies;
tau protein
Biomolecules 2016,6, 6; doi:10.3390/biom6010006 www.mdpi.com/journal/biomolecules

Biomolecules 2016,6, 6 2 of 28
1. Selective Overview of Major Discoveries on Tau Protein and Tauopathies
1.1. Neurofibrillary Tangles and Paired Helical Filaments
The Bavarian psychiatrist Aloysius (Alois) Alzheimer is credited with the first description of
the most characteristic pathological brain change—neurofibrillary tangles (NFT)—of a yet-unnamed
disease in a 51-year-old woman from Frankfurt am Main, who had developed dementia. That woman
was the first person to receive a diagnosis of the disease for which in 1910 Emil Kraepelin coined the
term Alzheimer’s disease (AD; which he wrongly, albeit cautiously, initially described as “presenile
dementia”). Her name was Auguste Deter and she had an early-onset dementia, comorbid with
psychotic features. As she became progressively worse, she had to be admitted to a psychiatric hospital
in November 1901 (where Alzheimer examined her for the first time), where she eventually died
in April 1906. Besides the already known “miliary foci” of extracellular deposits scattered over the
cerebral cortex (more commonly later called senile plaques, SP, or neuritic plaques, NP), by using a
newly developed silver staining method (20% water solution of silver nitrate, [
1
]) Alzheimer observed
degenerating cortical neurons with bundles of intracellular fibrils (neurofibrillary tangles, NFT) [2,3].
It was not until 1963 that with the help of electron microscopy, Kidd and Terry independently
reported NFT to be made up of abnormal filaments alternating between 15 (at their narrowest point)
and 30 nm (at their widest point) in width, with a half-periodicity of about 80 nm [
4
,
5
]. Because
it appeared that the two filaments were wound helically around one another, Kidd named them
paired helical filaments (PHF). Also found in NFT of AD, as a minority species, was the so-called
straight filament (SF), a filament about 15 nm wide that does not exhibit the marked modulation
in width shown by the PHF. Due to the fact that PHF were observed to be insoluble in denaturing
agents such as sodium dodecyl sulfate (SDS) and urea, despite significant efforts the structural and
molecular composition of PHF (and NFT) was not elucidated until the mid-1980s [
6
,
7
]. Morphological
studies of fragmentation patterns showed that the PHF actually consists of a left-handed helical
ribbon consisting of repeating symmetrical subunits. Using electron diffraction, Crowther and Wischik
were able to establish conclusively that the PHF is made up of a double helical stack of transversely
oriented C-shaped subunits, each of which has three domains. This structure precluded purely
descriptive models available to that point based on rearrangements of preformed cytoskeletal polymers
or protofilaments. They concluded that the structure was of a type that might arise from the de novo
assembly of a single structural subunit, the biochemical identity of which was then unknown. Later
studies showed that SF were composed of a similar structural subunit although with a slightly different
relative arrangement in the two types of filaments [8].
1.2. Tau Protein Isolation and Localization
Tau (tubulin-associated unit) protein was isolated from porcine brain extracts as a heat-stable,
highly soluble protein essential for microtubule (MT) assembly [
9
]. Following the initial discovery of
tau, two studies reported the process of tau purification and its physical and chemical properties [
10
,
11
],
including the ability of tau to become phosphorylated. In 1983, it was discovered that tau could be
phosphorylated at multiple sites by various protein kinases, including cyclic-AMP-dependent protein
kinases and casein kinase type-1 [
12
]. Further studies showed that tau is a phosphoprotein and that
phosphorylation negatively regulates its ability to stimulate MT assembly [13,14].
An immunohistochemical study that compared the localization of tau using the tau-1 antibody
(that recognizes all isoforms of tau, see below) with that of microtubule-associated protein 2 (MAP2)
and tubulin in human postmortem brain tissue demonstrated that tau protein was primarily localized
to axons [
15
]. Using the same tau-1 monoclonal antibody and electron microscopy with colloidal
gold-labeled secondary antibodies, tau was also found in very low amounts in astrocytes [
16
]
and oligodendrocytes [
17
], and this was confirmed by tau mRNA expression analysis in the
mouse brain [18].

Biomolecules 2016,6, 6 3 of 28
1.3. Tau in Neurofibrillary Tangles
The insolubility of PHF precluded biochemical characterisation of the repeating subunit that
makes up the structural core of the filament. What was required was a means of solubilising or releasing
the structural subunit as a protein band that could be visualised by gel electrophoresis and linking this
by immuno-electron microscopy to the PHF. Initial attempts based on relatively crude preparations
of NFT were unable to distinguish between proteins copurifying with NFTs due to trapping and
loose association within the dense filament bundles, and proteins derived from the structural core
of the PHF. In 1985 Brion and collaborators prepared tau and MAP2 proteins from the adult rat
brain using the microtubule assembly-disassembly method and their property of thermostability;
they then generated antisera against tau and MAP2 proteins using polypeptides extracted from
polyacrylamide gels after electrophoretic separation by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) [
19
]. Antisera were characterized by immunoblotting on purified
preparations of tau and MAP2 and found to react with their cognate antigens. These antisera were
then used for immunocytochemistry on tissue sections from control subjects and AD patients: the
anti-MAP2 antibody did not label NFT but the anti-tau antibody strongly immunolabelled NFT and
abnormal neurites around senile plaques, yielding an immunolabelling indistinguishable from the
one obtained with anti-PHF serum [
19
]. This work therefore established that tau protein was one of a
number proteins associated with NFTs both histologically and in crude NFT extracts. Neurofibrillary
tangles can be labeled histologically with antibodies against a variety of other neuronal proteins,
including vimentin, actin, ubiquitin, MAP2, and A
β
protein. In crude NFT preparations, isolated NFT
could be labeled with antibodies against MAP2, neurofilament, ubiquitin and tau [19–29].
The proof that tau protein contributes to the structural core of the PHF required preparation of
fractions highly in enriched in proteolytically stable PHF which retained the subunit structure of the
filament that had been characterised previously. These PHF were solubilised in formic acid and when
examined by SDS-PAGE gel electrophoresis were found to contain predominantly a 12-kD protein and
a corresponding dimer. Surprisingly, this protein was not recognised by an antibody raised against
tau protein. Conversely, a monoclonal antibody raised against the enriched core PHF preparations
(mAb 6.423, referred to as MN423) did not recognised purified tau protein [
30
–
33
]. Nevertheless,
MN423 was shown by immunogold electron microscopy to label the proteolytically stable core of
the PHF. Furthermore, a ligand related to primulin [
34
] was used to affinity label the 12-kD species.
This ligand, when bound covalently to biotin, was also shown by immunogold electron microscopy
to label proteolytically stable core PHF. Therefore, the provenance of the 12-kD protein from the
structural core of the PHF was unequivocally established by two independent approaches. Partial
amino acid sequences derived from this band were unrelated to any protein sequence known at that
time. However, when these were used to clone and sequence the corresponding cDNAs from a human
brain library, the predicted protein was found to be 352 amino acids in length and was found to have
extensive homology to the sequence of the mouse microtubule-associated protein tau isoform that had
just been published [
35
,
36
]. It was concluded that this protein must constitute the human homolog of
mouse tau, and that tau protein therefore must contribute to the structural core of the PHF, and was not
simply a loosely associated protein copurifying with NFT. These data were therefore able to explain the
earlier observations linking tau protein with NFT [
21
,
37
–
41
]. In the same year tau cDNA clones were
identified in the human fetal brain by flow sorting and spot-blot hybridization and later on assigned to
the microtubule-associated protein tau gene (MAPT) on the long arm of the chromosome 17 [42,43].
1.4. Tau Isoforms in the Central Nervous System
In their 1988 paper, Goedert and collaborators also mentioned that they had identified a second
form of tau, with sequence variation in the first repeat, and suggested that tau mRNA was undergoing
alternative splicing [
34
]. This second form was identical to the first, with the exception of an additional
insert of 31 amino acids in the repeat region. Upon sequencing of genomic clones, the extra repeat was
shown to be encoded by a separate exon, now known as exon 10. This work uncovered the existence

Biomolecules 2016,6, 6 4 of 28
of at least two types of tau isoforms in the human brain, with three repeats (3R tau) or four repeats
(4R tau) of a conserved tubulin-binding motif [
44
]. Sequencing of a large number of cDNA clones
revealed the existence of additional tau isoforms with two (29-N1, and 59 amino acid-N2) inserts in the
N-terminus region (due to alternative splicing of exons 2 and 3), in combination with both three and
four repeats (Figure 1). With the isoforms described previously, this gave a total of six human brain
tau isoforms ranging from 352 to 441 amino acids in length [
45
]. The primary sequence of the longest
tau isoform is shown in Figure 2. The most prominent expression of tau was observed during fetal
development, when only the shortest (referred to as fetal tau) isoform (N0R3) is expressed (352 amino
acids with molecular weight of 45 kDa), while the adult human brain expresses all isoforms with R4
to R3 ratio equal to 1 [
46
]. Relative amounts of N0, N1 and N2 tau isoforms are 37%, 54% and 9%,
respectively [
46
]. There is a further larger transcript of tau that encodes for a protein of 110 kDa with
an additional 254 amino acids in the N-terminal projection arm, but this protein is generally restricted
to the peripheral nervous system [47].
Biomolecules 2016, 6, 6 5 of 35
shown in Figure 2. The most prominent expression of tau was observed during fetal development,
when only the shortest (referred to as fetal tau) isoform (N0R3) is expressed (352 amino acids with
molecular weight of 45 kDa), while the adult human brain expresses all isoforms with R4 to R3 ratio
equal to 1 [46]. Relative amounts of N0, N1 and N2 tau isoforms are 37%, 54% and 9%, respectively [46].
There is a further larger transcript of tau that encodes for a protein of 110 kDa with an additional 254
amino acids in the N-terminal projection arm, but this protein is generally restricted to the peripheral
nervous system [47].
Figure 1. Chromosomal location of the gene and protein structure for the microtubule-
associated proteins tau, microtubule-associated protein 2 (MAP2) and MAP4. Tau exons 2,
3 and 10 are alternatively spliced, giving rise to six different mRNAs, translated in six
different tau isoforms. Tau isoforms differ by the absence or presence of one or two 29
amino acid inserts encoded by exon 2 (yellow) and 3 (orange) in the N-terminal part, in
combination with either three (R1, R3 and R4) or four (R1-R4) repeat regions in the C-
terminal part. The R2 repeat is encoded by exon 10. The longest 2N4R adult tau isoform
(2+3+10+) has 441 amino acids (aa), followed by 1N4R isoform of 412 aa (2+310+),
2N3R isoform of 410 aa (2+3+10), 0N4R isoform of 383 aa (2310+), 2N3R isoform of
381aa (2+310) and the shortest 0N3R isoform of 352 aa (2310). The single neuron-
specific promoter of MAPT gene has three binding sites for transcription factors and its
activity increases with axon initiation and outgrowth. The shortest tau isoform is the only
one expressed in the fetal brain (“fetal tau”), while expression of other isoforms begins
postnatally (for a review, see [48]). The MAP2 and MAP4 have comparable repeat domain
sequences in the C-terminus but differ from tau proteins by their longer N-terminal
projection arms.
Figure 1.
Chromosomal location of the gene and protein structure for the microtubule-associated
proteins tau, microtubule-associated protein 2 (MAP2) and MAP4. Tau exons 2, 3 and 10 are
alternatively spliced, giving rise to six different mRNAs, translated in six different tau isoforms.
Tau isoforms differ by the absence or presence of one or two 29 amino acid inserts encoded by exon
2 (yellow) and 3 (orange) in the N-terminal part, in combination with either three (R1, R3 and R4) or
four (R1-R4) repeat regions in the C-terminal part. The R2 repeat is encoded by exon 10. The longest
2N4R adult tau isoform (2+3+10+) has 441 amino acids (aa), followed by 1N4R isoform of 412 aa
(2+3
´
10+), 2N3R isoform of 410 aa (2+3+10
´
), 0N4R isoform of 383 aa (2
´
3
´
10+), 2N3R isoform of
381aa (2+3
´
10
´
) and the shortest 0N3R isoform of 352 aa (2
´
3
´
10
´
). The single neuron-specific
promoter of MAPT gene has three binding sites for transcription factors and its activity increases with
axon initiation and outgrowth. The shortest tau isoform is the only one expressed in the fetal brain
(“fetal tau”), while expression of other isoforms begins postnatally (for a review, see [
48
]). The MAP2
and MAP4 have comparable repeat domain sequences in the C-terminus but differ from tau proteins
by their longer N-terminal projection arms.

Biomolecules 2016,6, 6 5 of 28
Biomolecules 2016, 6, 6 6 of 35
Figure 2. Primary sequence of amino acids and probable secondary structure of the longest
tau isoform in the central nervous system. N1 and N2 denote the sequences encoded by
exons 2 and 3, respectively. R1 through R4 are microtubule-binding domains encoded by
exons 9–12, respectively. Domains with -sheet structure and -helical content are shown in
yellow and red, respectively.
Enriched preparations of PHF from extracts of homogenates from AD brains by using
N-lauroylsarcosine (sarkosyl) and 2-mercaptoethanol, after removal of aggregates by microfiltration,
sucrose density centrifugation and immunoblotting, revealed three tau bands of 60, 64, and 69 kDa ([49];
a minor fourth band of 72 kDa being described later by Mulot et al. [50]). This finding fitted well
with the previous findings obtained using monoclonal [51] and polyclonal tau antibodies [52].
Epitopes recognised by tau antibodies and phosphorylation sites on tau protein are shown in Figure 3.
In 1991, Lee and collaborators purified PHF using a method comparable to that of Greenberg and
Davis [53], and using protein chemical analysis claimed that they are made entirely of full-length
hyperphosphorylated tau protein [53].
In 1992, it was shown that, after dephosphorylation, the PHF-tau bands aligned with the recombinant
tau isoform mixture, indicating that PHF-tau consists of all six tau isoforms in a hyperphosphorylated
state [46]. These studies led to the widely quoted view that PHF are composed entirely of full-length
hyperphosphorylated tau protein. This view was challenged by the Wischik group who, using another
monoclonal antibody (mAb 7.51), also raised against enriched proteolytically stable core PHF and
recognizing all tau isoforms, showed that phosphorylated tau protein released from PHF preparations
by the sarkosyl method was not quantitatively related to the total PHF-tau protein content present in
these preparations, whether these were prepared with or without exogenous proteases [54,55]. Indeed
the proportion of full-length, phosphorylated tau protein was found biochemically to account for less
Figure 2.
Primary sequence of amino acids and probable secondary structure of the longest tau isoform
in the central nervous system. N1 and N2 denote the sequences encoded by exons 2 and 3, respectively.
R1 through R4 are microtubule-binding domains encoded by exons 9–12, respectively. Domains with
β-sheet structure and α-helical content are shown in yellow and red, respectively.
Enriched preparations of PHF from extracts of homogenates from AD brains by using
N-lauroylsarcosine (sarkosyl) and 2-mercaptoethanol, after removal of aggregates by microfiltration,
sucrose density centrifugation and immunoblotting, revealed three tau bands of 60, 64, and 69 kDa ([
49
];
a minor fourth band of 72 kDa being described later by Mulot et al. [
50
]). This finding fitted well with
the previous findings obtained using monoclonal [
51
] and polyclonal tau antibodies [
52
]. Epitopes
recognised by tau antibodies and phosphorylation sites on tau protein are shown in Figure 3. In 1991,
Lee and collaborators purified PHF using a method comparable to that of Greenberg and Davis [
53
], and
using protein chemical analysis claimed that they are made entirely of full-length hyperphosphorylated
tau protein [53].
In 1992, it was shown that, after dephosphorylation, the PHF-tau bands aligned with the
recombinant tau isoform mixture, indicating that PHF-tau consists of all six tau isoforms in a
hyperphosphorylated state [
46
]. These studies led to the widely quoted view that PHF are composed
entirely of full-length hyperphosphorylated tau protein. This view was challenged by the Wischik
group who, using another monoclonal antibody (mAb 7.51), also raised against enriched proteolytically
stable core PHF and recognizing all tau isoforms, showed that phosphorylated tau protein released
from PHF preparations by the sarkosyl method was not quantitatively related to the total PHF-tau
protein content present in these preparations, whether these were prepared with or without exogenous
proteases [
54
,
55
]. Indeed the proportion of full-length, phosphorylated tau protein was found
biochemically to account for less that 5% of total PHF-tau in the bulk PHF fraction prepared without
protease, and less than 15% in the sarkosyl PHF preparation. Although PHF isolated without protease
digestion can be immunolabeled by tau antibodies directed against phosphorylation-dependent
epitopes located in the N-terminal half of the molecule, this immunoreactivity is lost after proteolytic
removal of the fuzzy coat [
30
,
31
]. The fuzzy coat consists of the lengthy N-terminal portions of tau
molecules that cover the surface of the filaments and are readily sensitive to proteolytic digestion.

Biomolecules 2016,6, 6 6 of 28
Such digestion leaves intact the proteolytically stable core structure comprising the left-handed
helical or straight ribbon of repeated C-shaped subunits. In other words, the fuzzy coat comprising
phosphorylated tau does not contribute to the structural core of the PHF. Core PHF have a mass of
65 kD/nm, whereas 90% of PHF isolated without proteases is 77 kD/nm, with a further 10% having a
mass of 110 kD/nm [
31
]. The core PHF therefore accounts for ~85% of the mass of the filament, with a
variable addition of fuzzy coat material which contributes ~12 kD/nm.
Biomolecules 2016, 6, 6 7 of 35
that 5% of total PHF-tau in the bulk PHF fraction prepared without protease, and less than 15% in the
sarkosyl PHF preparation. Although PHF isolated without protease digestion can be immunolabeled by
tau antibodies directed against phosphorylation-dependent epitopes located in the N-terminal half of the
molecule, this immunoreactivity is lost after proteolytic removal of the fuzzy coat [30,31]. The fuzzy
coat consists of the lengthy N-terminal portions of tau molecules that cover the surface of the filaments
and are readily sensitive to proteolytic digestion. Such digestion leaves intact the proteolytically stable
core structure comprising the left-handed helical or straight ribbon of repeated C-shaped subunits. In other
words, the fuzzy coat comprising phosphorylated tau does not contribute to the structural core of the
PHF. Core PHF have a mass of 65 kD/nm, whereas 90% of PHF isolated without proteases is 77 kD/nm,
with a further 10% having a mass of 110 kD/nm [31]. The core PHF therefore accounts for ~85% of
the mass of the filament, with a variable addition of fuzzy coat material which contributes ~12 kD/nm.
Figure 3. Putative phosphorylation sites on tau protein and epitopes specific for major tau
antibodies. Red color denotes amino acids phosphorylated in AD brain, green in both AD
and normal brain, blue in normal brain, while black color means that those phosphorylation
sites have not been fully characterized yet. Tau antibodies specific for phospho-tau epitopes
are given in purple, while pink color denotes antibodies specific for non-phosphorylated
tau epitopes: Alz-50 (aa 2–10, aa 312342), 43D (aa 1–100), 77E9 (aa 185–195), 39E10
(aa 189–195), Tau-5 (aa 210–230), 5C7 (aa 267–278), Tau-1 (aa 195, 198, 199 and 202),
77G7 (aa 270–375), Tau-46 (aa 404–441), TauC-3 (tau cleaved on aa 421). Red—in the
AD brain; Green—in both the AD and the normal brain; Blue—in the normal brain;
Black—phosphorylation sites that have not been fully characterized yet; Purple—tau antibodies
specific for phospho-tau epitopes; Pink—tau antibodies specific for unphosphorylated tau epitopes.
Figure 3.
Putative phosphorylation sites on tau protein and epitopes specific for major tau antibodies.
Red color denotes amino acids phosphorylated in AD brain, green in both AD and normal brain,
blue in normal brain, while black color means that those phosphorylation sites have not been fully
characterized yet. Tau antibodies specific for phospho-tau epitopes are given in purple, while pink
color denotes antibodies specific for non-phosphorylated tau epitopes: Alz-50 (aa 2–10, aa 312
´
342),
43D (aa 1–100), 77E9 (aa 185–195), 39E10 (aa 189–195), Tau-5 (aa 210–230), 5C7 (aa 267–278), Tau-1
(aa 195, 198, 199 and 202), 77G7 (aa 270–375), Tau-46 (aa 404–441), TauC-3 (tau cleaved on aa 421).
Red—in the AD brain; Green—in both the AD and the normal brain; Blue—in the normal brain;
Black—phosphorylation sites that have not been fully characterized yet; Purple—tau antibodies
specific for phospho-tau epitopes; Pink—tau antibodies specific for unphosphorylated tau epitopes.
The tau species identified in the 12-kD species isolated from the PHF core were found to be
derived from a mixture of fragments originating from both 3- and 4-repeat isoforms, but restricted
to the equivalent of three repeats in length. There are two distinct species originating from the 4R
isoform of tau: the fragment derived from repeats 1, 2 and 3 and the fragment derived from repeats
2, 3 and 4. There is also a fragment derived from the 3R isoform (which lacks the second repeat),
comprising the equivalent of repeats 1, 3 and 4. Since all of these fragments are restricted to the
equivalent of three repeats in length, they all have identical gel mobility [
56
]. Therefore, the PHF is
composed of a structural core of repeating transverse C-shaped subunits. The only tau protein found
within the subunit structure is restricted to the repeat domain of the tau molecule. The contribution
of phosphorylated tau is variable and restricted to the fuzzy outer coat of the PHF, accounting for
approximately one in seven of the tau molecules found in the PHF [57].

Biomolecules 2016,6, 6 7 of 28
1.5. Functions of Tau Protein
Tau protein is most abundantly expressed in axons of central nervous system neurons [
13
] but
can also be found in the somatodendritic compartment of neurons, oligodendrocytes, and non-neural
tissues [
58
]. Probably the most important role of tau protein is to promote assembly and stability
of MT [
7
,
8
], although this function is complemented by other MAP (especially by MAP1B), as tau
knockout mice are viable, fertile, and relatively normal, with no signs of neurodegeneration. Also,
knockdown of tau with small interfering RNA does not kill primary neurons in culture or prevent
axon formation [
59
]. Additionally, MAP1B is probably more important for MT stability than tau itself
because knockout of MAP1B results in abnormal brain development and early death, and concurrent
knockout of both MAP1B and MAPT worsens the phenotype [60].
The most common post-translational modifications of tau proteins are phosphorylation and
O-glycosylation [
61
]. Phosphorylation changes the shape of tau molecule and regulates its biological
activity. Most of the phosphorylation sites are on Ser-Pro and Thr-Pro motives, but a number of sites
on other residues have also been reported [
62
,
63
]. The majority of tau-based therapeutic strategies
against neurodegeneration have focused on modulating tau phosphorylation, given that tau species
present within NFT are hyperphosphorylated. O-glycosylation is characterized by the addition of
an O-linked N-acetylglucosamine (O-GlcNAc) on Ser or Thr residues in the vicinity of Pro residues.
It is presumed that glycosylation may have a role in subcellular localization and degradation of tau
proteins [
64
]. The recent discovery that tau is also modified by acetylation requires additional research
to provide greater insight into the physiological and pathological consequences of tau acetylation [
65
].
Tau protein can be divided into two main functional domains: the basic MT binding domain
(towards the C-terminus) and the acidic projection domain (towards the N-terminus) [
66
]. The MT
binding domain regulates the rate of MT polymerization through highly conserved repetitive domains
R1–R4 encoded by exons 9–12 [
36
]. Adult tau isoforms with 4R (R1–R4) are about 40-fold more
efficient at promoting MT assembly than the fetal isoform that is lacking exon 10 and thus having
only 3R (Figure 1) [
67
]. The absence of expression of the R1–R2 inter-repeat region during fetal
development allows for the cytoskeletal plasticity required of growing immature neurons and their
elongating axons [
64
]. Apart from binding to MT, the repeat domains of tau also bind to tubulin
deacetylase, histone deacetylase 6 (HDAC6) [
68
] and apolipoprotein E (apoE, more with the
ε
3 than
the ε4 isovariant [69]).
The projection domain is so called because ultrastructurally it appears as a filamentous “arm”
projecting from the wall of the MT. In recent years, many hitherto unknown binding partners
of the projection domain have been identified. The projection domain of tau may be involved
in cell signaling that occurs through the interaction with Lck, Fgr and cSrc (Src-family kinases),
growth factor receptor-bound protein 2 (Grb2), phospholipase C-
γ
[
70
], phosphatidylinositol and
phosphatidylinositol bisphosphate [
71
,
72
], peptidyl-prolyl cis/trans isomerase Pin 1, and many others
(for review see [
73
]), making them potential therapeutic targets in tauopathies [
74
]. In synapses,
the projection domain of tau interacts with protein kinase Fyn (plays an important role during
myelination [75]), postsynaptic density protein 95 (PSD-95) [76], and N-methyl-D-aspartate receptors
(NMDAR). Tau knockout mice show that tau is essential for NMDA-dependent long-term potentiation
(LTP) and
α
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-dependent long-term
depression [
77
–
79
]. The function of tau protein in the response to heat stress in the cell is also worth
noting. When the heat stress occurs, tau protein binds to DNA and enhances DNA repair [
80
].
An additional “knot” of tau being entangled in epigenetic landscape of neurodegeneration comes from
the finding that by acting as a HDAC6 inhibitor, tau is being indirectly involved in both (dys)regulation
of transcriptional activity and impairment of autophagic clearance by the ubiquitin proteasome
system [81,82].

Biomolecules 2016,6, 6 8 of 28
1.6. Amyloid Cascade Theory
The so-called “cholinergic hypothesis of AD” [
83
,
84
] dominated the late 1970s and early 1980s,
and the “calcium hypothesis” in the late 1980s. After the milestone discovery that cerebrovascular
amyloid and NP are composed of A
β
(as they shared the same antigenic determinants; [
85
,
86
]) in
both AD and Down syndrome [
87
], and that the V717I missense “London” mutation in the amyloid
precursor protein gene (APP) on chromosome 21 was found to be causally related to the early-onset
autosomal-dominant familial AD [
88
], Hardy and colleagues [
89
,
90
] proposed the “amyloid cascade
hypothesis”, which has become a dominant driver of AD research ever since. According to the
amyloid theory, excessive production of A
β
via serial cleavage of the larger amyloid precursor protein
(APP) molecule by
β
-secretase (
β
-site APP cleaving enzyme, BACE, encoded by the BACE1 gene)
and
γ
-secretase (multiprotein complex now known to consist minimally of four individual proteins:
presenilin, nicastrin, anterior pharynx-defective 1, APH-1, and presenilin enhancer 2, PEN-2; [
91
–
93
]),
is the key pathological event which drives all other pathological changes (astrocytosis, microglial
activation, neuronal death, synaptic loss, and the development of NFTs and dementia) not only
in early-onset familial cases but also in late-onset, sporadic cases of AD. In 1987 Goldgaber and
collaborators isolated APP and localized its gene to chromosome 21 [
94
]. It should be noted here that
the first APP mutation discovered was actually the G to C mutation at codon 693 (APP E693Q) that
was not associated with AD, but rather with hereditary cerebral hemorrhage with amyloidosis—Dutch
type (HCHWA-D; [
95
,
96
]). Interestingly enough, out of four other known mutations within the A
β
part of APP (exons 16 and 17) two also cause fatal hemorrhage due to amyloid angiopathy (APP
C692G-Flemish and APP E693K-Italian), while only rare “Arctic” (APP E693G) and Osaka (APP E693
∆
)
mutations cause early-onset AD (EOAD). The well-known fact that many families exist in which
AD has an early onset (before age of 60) and is inherited in an autosomal dominant manner [
97
]
could not be explained by a very small number of AD families in which APP mutations were found.
This question was resolved in part by the discovery of mutations in the presenilin 1 gene (PSEN1) on
chromosome 14 [
98
,
99
], and its homologous presenilin 2 gene (PSEN2) on chromosome 1 [
100
,
101
].
These mutations of PSEN genes further strengthened the amyloid theory, but the pathogenesis of AD
remained elusive. Further research showed that PSEN1 and PSEN2 are part of the
γ
-secretase complex,
which cleaves APP at several points resulting in A
β
of various lengths: the lengths associated with
AD are 40 and 42 amino acids long with A
β42
more likely to aggregate to form SP in the brain than
A
β40
. All PSEN mutations lead to an increase in the A
β42
:A
β40
ratio, although the total quantity of
A
β
produced remains constant [
102
,
103
]. This can come about by various effects of the mutations
of
γ
-secretase. Presenilins are also implicated in the processing of notch [
104
,
105
], an important
developmental protein (mice that have PS1 knocked out die early in development from developmental
abnormalities similar to those found when notch is disrupted, [
106
]). APP can also be cleaved by
α
-secretases such as a disintegrin and metalloproteases domain 10 (ADAM10) and tumor necrosis
factor alpha (TNF-
α
) converting enzyme (TACE), although this cleavage does not result in A
β
but
instead generates APPs-α, which are thought to be neuroprotective [107].
Collectively, the genetic etiology of AD is very complex: early-onset AD (less than 5% of cases) is
often familial (fAD) with autosomal dominant and fully penetrant inheritance and can be caused by
any of more than 200 pathogenic mutations in APP (33 mutations, duplication), PSEN1 (185 mutations)
and PSEN2 (13 mutations; http://www.molgen.ua.ac.be/ADmutations). Most AD cases (over 95%)
however are sporadic, late-onset (sAD, LOAD) and have less evident genetic components. The
ε
4 variant of the gene encoding apolipoprotein E (APOE) is known to confer increased risk for
LOAD [
108
,
109
] with partial penetrance. Based on 320 meta-analyses of 1395 studies in which 695 genes
and their 2973 polymorphisms have been tested as late-onset AD candidate genes, over 30 yielded
positive evidence for association. The number one gene is APOE, with a Bayes factor (BF) > 50. Using
APOE genotype
ε
3/
ε
3 as a neutral benchmark for comparison, individuals with a single copy of the
ε
4 allele manifest a 5 fold increased chance of developing LOAD, while those with two copies have
an estimated 20 fold increased risk [
110
]. It seems that different APOE alleles are not associated with

Biomolecules 2016,6, 6 9 of 28
an increase in A
β
production, but with a reduced ability to clear A
β
from the brain [
111
,
112
]. This
may be related to decreased production of A
β
auto-antibodies in AD subjects [
113
]. The next nine
genes with the highest association with LOAD are: BIN1 (BF = 23.4) that encodes several isoforms
of a nucleoplasmic adaptor protein, one of which was identified as MYC-interacting protein, CLU
(BF = 20.1) that encodes apolipoprotein J, ABCA7 (BF = 18.8) for ATP-binding cassette, subfamily A
[ABC1], member 7, CR1 (BF = 18.1) for complement component receptor 1, PICALM (BF = 17.3) for
phosphatidylinositol-binding clathrin assembly protein, MS4A6A (BF = 8.7), CD33 (BF = 7.7) for a
transmembrane receptor expressed on cells of myeloid lineage-cluster of differentiation 33, MS4A4E
(BF = 6.9) coding for protein membrane-spanning 4-domains, subfamily A, member 4E, and CD2AP
(BF = 6.6) that codes for a scaffolding molecule that regulates the actin cytoskeleton (according to
www.alzgene.org accessed on 11 February 2015). Genetic variants of all of these genes have a relatively
minor influence on AD progression when altered [
114
] and their influence on the development and
course of sAD remains largely unknown [
115
–
117
]. Most recently, rare mutations of TREM2 [
118
]
and PLD3 [
119
] have also been discovered to confer a much larger increase in risk for LOAD than the
aforementioned common sequence variants [120].
1.7. Staging of Tau Pathology
During the 1990s, the significance of tau pathology for neurodegenerative diseases, in particular
for AD, remained in the shadow of the amyloid theory. However, as the distribution pattern and overall
quantity of A
β
turned out to be of limited significance for pathological staging of AD progression
and symptom severity, and after detailed studies of the maturation and distribution of NFTs showing
correlation with degree of cognitive decline and memory impairment in AD, Braak and Braak proposed
a neuropathological staging of the gradual deposition of abnormal tau within vulnerable neurons
in brain areas in the form of either NFT or neuropil threads (NT). At first they used classical silver
staining [
66
] and later immunohistochemical staining for hyperphosphorylated tau using antibody
AT8 [
67
]. The finding that NFT provide a better association with cognitive impairment was confirmed
by other researchers [
121
,
122
], supporting a significant role for tau pathology in the disease. The Braak
staging system classified the topographic progression of AD neurofibrillary degeneration into six
stages, spreading from the transentorhinal region to the hippocampal formation (initial stages I and II,
which clinically correlate with subjective or objective impairment of memory for recent events and
mild spatial disorientation, but with preservation of general cognitive functioning with or without
minimum impairment of activities of daily living), then to the temporal, frontal, and parietal neocortex
(intermediate stages III and IV, which correlate with impaired recall, delayed word recall and word
finding difficulties, disorientation in time and space, and impaired concentration, comprehension and
conceptualization among other symptoms of dementia), and finally to unimodal and primary sensory
and motor areas of the neocortex (late stages V and VI, which roughly correlate with disturbances in
object recognition, and other perceptual and motor skills).
Besides the fact that Braak and collaborators showed that AD-related pathology proceeds in
strictly defined stages, based on the notion that NFT evolve from an accumulation of abnormal
tau without PHF formation (described as the “pre-tangle” stage, [
123
]) they also proposed that
abnormal phosphorylation is a crucial step leading to the formation of both soluble and insoluble tau
filaments [
124
], that neuronal damage in AD actually starts many years before any clinical symptoms
and signs and that, unlike A
β
, the distribution of tau pathology is associated with the clinical
progression of AD. In contrast to the amyloid cascade hypothesis of AD, which implies that tau
pathology is a secondary, downstream phenomenon, the neuropathological findings of Braak and
collaborators have fueled a significant controversy concerning the importance or contributions of A
β
burden in producing damage compared to that caused by tau pathology. Additionally, in AD, the
pathological A
β
and tau proteins mutually interact and are influenced by many other contributors,
such as inflammatory [
125
], vascular, and environmental factors, as well as compensatory neuroplastic

Biomolecules 2016,6, 6 10 of 28
responses to counteract neural injury associated with neurodegenerative processes [
126
], all of which
may promote cognitive and behavioral decline.
1.8. Mutations in MAPT Gene and Tauopathies
In the late 1980s and early 1990s, evidence implicating tau pathology in neurodegenerative
diseases other than AD began to emerge. As early as 1986, Pollock and colleagues reported that the
filamentous aggregates in Pick’s disease (now part of the group of disorders classes as frontotemporal
lobar degeneration, FTLD, and called frontotemporal dementia, FTD), progressive supranuclear palsy
(PSP) and AD shared the antigenic determinants of tau [
127
]. While hyperphosphorylation of tau is a
feature common to all of these diseases, unlike AD, they lack significant A
β
and
α
-synuclein pathology.
However, biochemical differences in the tau isoforms isolated from preparations of the pathological
filaments in various tauopathies were observed. All six tau isoforms are present in sarkosyl extracts
in equal ratios of R3 and R4 isoforms is observed in class I tauopathies, which are biochemically
characterized by tau triplets of 60, 64 and 69 kDa, and additional minor bands of 72/74 kDa.
Such a profile is characteristic for AD, some cases of frontotemporal dementia and parkinsonism
linked to chromosome 17 (FTDP-17), Niemann-Pick disease type C, Down syndrome and dementia
pugilistica [
128
]. On the other side, sarkosyl extracts from the filaments of PSP [
129
], corticobasal
degeneration (CBD; [
130
]), argyrophilic grain disease (AgD; [
131
]), and some cases of FTDP-17, contain
tau protein that separates as doublets of 64 and 69 kDa and are predominantly composed of tau
isoforms with 4R (class II tauopathies), whereas sarkosyl extracts from filaments of Pick’s disease
are characterized by the presence of pathological tau doublets of 60 and 64 kDa and contain mainly
3R tau isoforms (class III tauopathy). Class IV tauopathy is represented by a single neurological
disorder—myotonic dystrophy type I (DM1) or Steinert’s disease, in which a major insoluble tau band
of 60 kDa, and minor 64 and 69 kDa bands have been identified [
48
,
61
,
76
,
128
,
132
,
133
]. Despite the fact
that these studies showed the filaments contain tau, they did not provide much direct information
about the relevance of tau dysfunction and filament formation in the neurodegenerative disease process.
Although tau involvement in neurodegenerative diseases other than AD attracted wide attention,
genetic evidence linking dysfunction of tau protein to neurodegeneration and dementia had been
missing. In 1994, Wilhelmsen and colleagues reported linkage of an autosomal dominantly inherited
form of FTD with parkinsonism and amyotrophy (disinhibition-dementia-parkinsonism-amyotrophy
complex, DDPAC) to chromosome 17q21.2, the region that contains the MAPT gene [
134
]. In 1997,
Spillantini and colleagues first used the term tauopathy to describe “multiple system tauopathy with
presenile dementia” (MSTD), where tau filaments contain 4R isoforms in absence of 3R tau [
135
].
In parallel, Murrell and colleagues showed that the genetic defect in MSTD mapped to chromosome
17q21-22 [
136
]. At that time, 13 kindreds were considered to have sufficient evidence of linkage to be
included in what was then named “frontotemporal dementia and parkinsonism linked to chromosome
17” (FTDP-17; [
137
]). The exclusive presence of 4R tau in the MSTD filaments naturally led to an
examination of the isoform composition of the pool of soluble tau and its findings suggested that
increased splicing of exon 10 of the MAPT gene might be the cause of familial MSTD. Upon DNA
sequencing, a guanine (G) to adenine (A) transition at position +3 of the intron following exon 10 was
found, which segregated with disease [
138
]. At that time, an additional eight mutations in the MAPT
gene had been reported by two other groups: Poorkaj et al. reported two exonic mutations (P301L and
V337M) in two families with FTDP-17 [
139
], while Hutton et al. reported six different mutations in
10 families: three of these mutations (G272V, P301L and R406W) were missense mutations in exons,
while the other three were in the 5
'
splice site of exon 10 [
140
]. Later that year, missense mutations
were shown to reduce the ability of tau to promote microtubule assembly [141,142].
The discovery of these and other subsequent mutations in the MAPT gene finally confirmed that
molecular tau pathology can give rise to neurodegeneration in the absence of A
β
changes and that tau
is in a central position as a key pathological component (leading from normal, soluble tau to abnormal,
filamentous tau which causes neurodegeneration and dementia) across many neurodegenerative

Biomolecules 2016,6, 6 11 of 28
states and disorders, either through mutations in the MAPT gene or the effects of upstream stressors
such as A
β
or oxidative damage (for review, see [
143
]). Why particular neurons are susceptible to
the buildup of misfolded tau and tau aggregation remained unanswered, leading to much research
activity on tau, with the development of transgenic animals and cell lines to model the effects of
expressing disease-related mutations of the MAPT gene. The level of biochemical diversity and,
consequently, complexity of tau pathology is perhaps best illustrated by the simple fact that specific
MAPT mutations are associated with specific forms of FTD; in contrast, the very same mutation (such
as TAU P301L) can apparently lead to either CBD or FTDP-17 in the same family, suggesting that other
factors (genetic, epigenetic, environmental) may influence which neurons are affected and when this
occurs. Several other findings have further emphasized the importance of tau in neurodegeneration
(reviewed in [
144
]). A PSEN1 mutation causes a Pick’s disease phenotype including FTD tau pathology
without deposition of A
β
[
145
]; some MAPT single nucleotide polymorphisms have also been linked to
sporadic Parkinson’s disease (PD, [
146
]); and retarded axonal extension in tau-deficient hippocampal
neurons may be due to reduced MT transport by lack of tau-mediated regulation of motor protein
activities [147].
2. Tau Protein Pathological Changes in Primary and Secondary Tauopathies
2.1. Mechanisms
As mentioned earlier, compelling evidence that tau malfunction or dysregulation alone can be
sufficient to cause neurodegeneration came in 1998 from the identification of mutations in the MAPT
gene on chromosome 17 that causes frontotemporal dementia with parkinsonism (FTDP-17) [
140
],
making cytoskeletal abnormalities a pivotal mechanism in neurodegeneration in AD (mutations in
the MAPT gene cause primary tauopathies, while AD is the most important secondary tauopathy
with the MAPT gene itself not being mutated) [
143
,
148
]. More specifically, abnormal phosphorylation,
aggregation, and proteolysis of the tau protein in a “pre-tangle” stage of neurofibrillary degeneration
(Figure 4) has been neuropathologically documented to be an early and crucial event in the
pathogenesis of AD, but also other sporadic tauopathies, such as AgD [
131
] and PSP. Historically, NFT
were considered indicators of cell death, particularly given that since 1995 they have been consistently
shown to correlate well with the severity of dementia in AD, in contrast to A
β
plaque deposition does
not [
122
]. However, which variety of tau is the most toxic (aggregated misfolded/fibrillar, soluble
hyperphosphorylated/mislocalized, or both) and whether that toxicity represents a gain or loss of
function remains an unanswered question. As there is little direct evidence that tau fibrils themselves
are toxic, the hypothesis that soluble oligomeric forms of tau are more toxic to neuronal and synaptic
function is increasingly gaining favor. The formation of NFT may actually protect neurons acutely
from the effects of toxic soluble tau, as shown by Kopeikina and collaborators [149].
The tau fragment first isolated from the PHF core is approximately 100 amino acids in length.
Its N-terminus was defined by sequence analysis [
30
,
56
], and its C-terminus was defined by epitope
mapping of MN423. Immunoreactivity was shown to depend on a specific C-terminal trunctation
at Glu391 [
33
,
150
]. Thus, the N-termini of the tau fragments found in the proteolytically stable
structural core of the PHF are located 15-residues C-terminal to the start of the repeats, and have
a characteristic C-terminal truncation at position Glu391 which is 15-residues C-terminally to the
end of the repeats [
151
]. These features explained the paradox noted earlier, namely that tau protein
isolated from the core of the PHF is not necessarily recognized by anti-tau antibodies (if these are
directed against epitopes located in the N- or C-terminal portions of the molecule, whether or not
phosphorylated), and the monoclonal antibody raised against the PHF core does not recognized
normal full-length tau protein [30].

Biomolecules 2016,6, 6 12 of 28
Biomolecules 2016, 6, 6 14 of 35
Figure 4. The sequence of cytoskeletal changes due to the pathology of tau protein divided
into three stages: pre-tangle (pre-NFT) stage, and intraneuronal and extraneuronal stages. See
text and Figure 5 for details.
Figure 5. Diagram showing sites for potential cleavage of tau protein. The sequential cleavage
of the tau protein leads to the formation of the tau protein fragment from the microtubule-
binding repeat region (see text and Figure 4). Tau cleavage is more likely to take place
while protein is unbound to microtubules, either aggregated to itself or associated with
proteins other than tubulin. PSA = puromycin-sensitive aminopeptidase.
Figure 4.
The sequence of cytoskeletal changes due to the pathology of tau protein divided into three
stages: pre-tangle (pre-NFT) stage, and intraneuronal and extraneuronal stages. See text and Figure 5
for details.
Biomolecules 2016, 6, 6 14 of 35
Figure 4. The sequence of cytoskeletal changes due to the pathology of tau protein divided
into three stages: pre-tangle (pre-NFT) stage, and intraneuronal and extraneuronal stages. See
text and Figure 5 for details.
Figure 5. Diagram showing sites for potential cleavage of tau protein. The sequential cleavage
of the tau protein leads to the formation of the tau protein fragment from the microtubule-
binding repeat region (see text and Figure 4). Tau cleavage is more likely to take place
while protein is unbound to microtubules, either aggregated to itself or associated with
proteins other than tubulin. PSA = puromycin-sensitive aminopeptidase.
Figure 5.
Diagram showing sites for potential cleavage of tau protein. The sequential cleavage of
the tau protein leads to the formation of the tau protein fragment from the microtubule-binding
repeat region (see text and Figure 4). Tau cleavage is more likely to take place while protein is
unbound to microtubules, either aggregated to itself or associated with proteins other than tubulin.
PSA = puromycin-sensitive aminopeptidase.

Biomolecules 2016,6, 6 13 of 28
The characteristic N- and C-terminal truncations found in the core PHF-tau unit are exactly
3-repeats apart, but shifted by 15-residues with respect to the N- and C-terminal extent of the normal
repeat domain. This feature was shown to represent the footprint of a pathological tau-tau binding
interaction, which can occur through the repeat domain of the tau molecule and which locks it into a
characteristic proteolytically stable configuration. The Glu391 trunctation could be reproduced
in vitro
after binding of full-length tau to a fragment terminating at Ala390 and lacking immunoreactivity to
MN423 [
152
]. Surprisingly, when the bound complex was taken through repeated cycles of digestion
with protease and re-incubation of full-length tau, N-terminal tau immunoreactivity was eliminated
in every cycle, whilst a progressive build-up of Glu391 immunoreactivity detected by MN423 was
observed. Thus, the repeat domain of tau is able to catalyze and propagate the conversion of normal
soluble tau into the aggregated and truncated oligomeric form in a cell-free setting.
The same phenomenon has recently been demonstrated within the physiological milieu of the cell.
Although the repeat domain fragment is highly toxic when expressed in cells, cells can be maintained
provided expression levels remain very low, where it acts as a latent primer or seed. When such
cells were co-transfected with inducible, full-length 4R tau, it was possible to induce tau aggregation
and template-directed truncation of full-length tau in a controlled manner [
153
]. The neo-fragment
generated in a concentration-dependent manner with respect to induced full-length tau proved to be
the core tau unit itself. Thus the repeat domain has the ability to define a template-directed truncation
of full-length tau to reproduce and amplify the proteolytically stable species characteristic of the PHF
core in AD recruiting normal tau in the process. Importantly, the process does not require abnormal
phosphorylation, or any other post-translational modification once it has been initiated. The same
phenomenon has been reported independently in transgenic rat studies expressing a truncated tau
species containing the repeat domain [
154
,
155
]. Insoluble tau aggregates were found to form in the
brain consisting of both transgenic human truncated tau and endogenous rat tau in a 1:1 ratio. A further
suprising feature in the cell-model was the demonstration that when full-length tau is induced in
the presence of the truncated tau primer, it is unable to bind normally to microtubules, but instead is
preferentially directed to the aggregation/truncation pathway. In contrast, full-length tau expressed
in the absence of the truncated tau primer showed normal tubulin binding [
153
]. These results are
consistent with that the pathological tau-tau binding affinity through the repeat domain is higher
than the normal tau-tubulin binding affinity [
57
]. Thus, it is unnecessary to invoke pathological
phosphorylation as a primary mechanism to account for loss of normal microtubule binding in AD.
Rather, the almost complete redistribution of the tau protein pool from soluble/tubulin-bound to
insoluble/aggregated that occurs in AD is simply a kinetic consequence of the properties of the
pathological tau-tau binding interaction of the repeat domain.
The truncated core tau unit of the PHF has also been termed “the F3 fragment” (Figure 5) [
156
].
The F3 fragment in the oligomeric state is resistant to cytosolic proteases and may therefore be
transported unchanged to axon terminals where it may not only damage synapses, but from where it
may propagate between neurons either trans-synaptically or by exosomes, thus initiating the same
neurofibrillary cascade in previously healthy neurons [
157
]. As recently reviewed by Jadhav and
collaborators [
76
], several factors point strongly towards a prominent role of presynaptic tau protein in
mediating synaptic pathology, including (1) cognitive decline that best correlates with synaptic loss
and synaptic failure; (2) synapse loss in parallel with NFT formation and occurring in the same regions
in AD brains; and (3) higher NFT count associated with lower levels of presynaptic proteins in AD. In a
prospective study three different synaptic protein (synaptophysin, SNAP-25 and syntaxin) were found
to be progressively in neocortex at Braak stages III-VI [
158
], NFT-bearing neurons demonstrating,
for example, a 35%–57% reduction in synaptophysin mRNA in AD brain [
159
]. Last but not least,
synaptic deficits are observed in FTLD [
160
], PSP, and Niemann-Pick disease type C [
161
], which are
all independent of any A
β
pathology. Moreover, tau proteomic changes are also confirmed in several
tau transgenic models (for review, see [76]).

Biomolecules 2016,6, 6 14 of 28
Tau fragments are also able to propagate between neurons trans-synaptically, causing the spread
of neurofibrillary degeneration to post-synaptic neurons [
162
]. In this case, mutations in the APP,
PSEN1 and PSEN2 genes in familial AD only initially compromise endosomal-lysosomal processing
and mitochondrial metabolism by altering A
β
clearance thus activating caspases responsible for tau
cleavage or providing seeding factors required to nucleate pathological aggregation of tau protein
through the repeat domain.
2.2. Seeding and Spreading of Tau Proteins
Tau can be directly involved in the spread of AD pathology to neighbouring neurons. However,
direct evidence for molecular mechanisms supporting this hierarchical progression (“prion-like
behaviour of misprocessed tau”) has remained elusive [
163
]. The most recent data obtained indicate
that tau pathology indeed may be induced and propagated after the injection of tau oligomers or
aggregates in either wild-type or mutated MAPT transgenic mice [
164
], and that tau aggregates can
be transferred from cell to cell
in vitro
[
164
,
165
] and
in vivo
[
166
,
167
]. These new findings suggest
that suppressing the spread of tau oligomers could be a target for development of disease-modifying
therapeutics for AD and other tauopathies, although further studies are needed to determine whether
pathologic tau oligomers spread trans-synaptically or by exosomes. Additionally, it was proved
that antibodies blocking tau aggregate seeding improve cognition
in vivo
[
168
]. In the case of soluble
monomeric or small oligomeric tau protein, the endocytosis appears to be clathrin-dependent (reviewed
in [
169
]). In contrast, larger aggregates of tau could bind heparin in the extracellular matrix and be
internalized through macropinocytosis [
170
]. As a result of exocytosis and endocytosis, the spreading
of tau can occur in various neurodegenerative diseases (tauopathies) including AD. Three plausible
mechanisms of tau spreading are shown schematically in Figure 6. Additionally, it appears that
microglial cells may facilitate tau propagation by phagocytosis and exocytosis of tau protein [
171
].
Avila et al. (2015) have speculated on how the top six LOAD risk genes (APOE,BIN1,CLU,ABCA7,
CR1 and PICALM), that all interact with tau, may be involved in the transmission of tau [172].
In one of our previous pilot studies, we observed evidence suggesting the spreading of tau
pathology in the brains of patients who suffered from mild cognitive impairment (MCI) with antibodies
specific for phospho-tau epitopes Ser202 and Thr205 (AT8), Ser396 (AD2) and PHF core (MN423).
In early MCI, only AT8 immunoreactivity was observed in the neurons of the superficial entorhinal
cortex layers and temporal isocortex. In more advanced MCI cases, we detected both AT8 and AD2
immunoreactivity in these neurons. Immunoreactivity to MN423 in layers II-III of the entorhinal and
temporal isocortical neurons was observed only in advanced MCI and early AD patients. Concurrently,
AT8 and AD2 immunoreactivity was found in neurons of layers II, III and V of the entorhinal cortex
and AT8-immunoreactive pyramidal neurons were already present in layers III and V of the temporal
isocortex. As an increase in MN423 immunoreactivity positively correlated with Braak stages, these
findings support the concept of Wischik and collaborators [
157
] that cycles of proteolytic removal of N-
and C-termini of tau are followed by stepwise binding of further tau in an autocatalytic process and
that the repeat domain of tau is able to catalyse and propagate the conversion of normal soluble tau
into accumulations of the aggregated and truncated oligomeric form.

Biomolecules 2016,6, 6 15 of 28
Biomolecules 2016, 6, 6 17 of 35
speculated on how the top six LOAD risk genes (APOE, BIN1, CLU, ABCA7, CR1 and PICALM), that
all interact with tau, may be involved in the transmission of tau [172].
In one of our previous pilot studies, we observed evidence suggesting the spreading of tau
pathology in the brains of patients who suffered from mild cognitive impairment (MCI) with
antibodies specific for phospho-tau epitopes Ser202 and Thr205 (AT8), Ser396 (AD2) and PHF core
(MN423). In early MCI, only AT8 immunoreactivity was observed in the neurons of the superficial
entorhinal cortex layers and temporal isocortex. In more advanced MCI cases, we detected both AT8
and AD2 immunoreactivity in these neurons. Immunoreactivity to MN423 in layers II-III of the
entorhinal and temporal isocortical neurons was observed only in advanced MCI and early AD
patients. Concurrently, AT8 and AD2 immunoreactivity was found in neurons of layers II, III and V of
the entorhinal cortex and AT8-immunoreactive pyramidal neurons were already present in layers III
and V of the temporal isocortex. As an increase in MN423 immunoreactivity positively correlated with
Braak stages, these findings support the concept of Wischik and collaborators [157] that cycles of
proteolytic removal of N- and C-termini of tau are followed by stepwise binding of further tau in an
autocatalytic process and that the repeat domain of tau is able to catalyse and propagate the conversion
of normal soluble tau into accumulations of the aggregated and truncated oligomeric form.
Figure 6. Schematic representation of three different ways of anterograde spreading of tau
aggregates by endocytosis, macropinocytosis, and exosomes.
2.3. Therapeutic Approaches Targeting Tau Protein Processing in Tauopathies
A number of neuroprotective strategies have been proposed based on the phosphorylation theory of
tau pathology (Figure 7).
Figure 6.
Schematic representation of three different ways of anterograde spreading of tau aggregates
by endocytosis, macropinocytosis, and exosomes.
2.3. Therapeutic Approaches Targeting Tau Protein Processing in Tauopathies
A number of neuroprotective strategies have been proposed based on the phosphorylation theory
of tau pathology (Figure 7).
Biomolecules 2016, 6, 6 18 of 35
Figure 7. Diagram showing potential neuroprotective strategies to reduce tau aggregates.
See text for details.
(1) MT-stabilizing agents, which as an approach does not address the accumulation of toxic
tau aggregates.
(2) Modulation of tau phosphorylation has been shown to prevent motor impairments in tau
transgenic mice [173]. Green coffee, a non-toxic small molecule, found to be an inhibitor of protein
phosphatase 2A methylesterase, was shown to improve cognitive and motor performance in mouse
models with tau pathology [174].
(3) A different approach, which does not depend on the phosphorylation theory, is based on
selective inhibition of pathological tau aggregation [157]. The problem in identifying suitable tau
aggregation inhibitors (TAI) is that most assays for tau aggregation are based on fibril formation
(which require relatively high concentrations of tau, tau constructs limited to the MT binding region
with the motif necessary for fibril formation, and a facilitator of aggregation, such as heparin). If
compounds are selected that dissociate preformed large aggregates into smaller (still toxic) oligomers
then they may be detrimental. In these assays, fibril formation is measured by a shift in fluorescence of
an intercalating reporter dye binding to -sheet structures within tau fibrils.
(4) A more promising approach may be to target tau oligomers, whether these are intracellular or
extracellular. Purified tau oligomer species have been demonstrated to be neurotoxic (they directly
impair synaptic function and long-term potentiation, LTP) in vitro in a dose-dependent manner.
Extracellular tau levels measured in AD are more than four orders of magnitude lower than
intracellular tau concentrations, and as such may represent a more amenable pharmacological target.
(5) A quite different strategy is to target tau clearance—e.g., by rapamycin that induces
macroautophagy [175], inhibitors of Hsp90 chaperone protein that binds to misfolded proteins or by
immunotherapeutic approaches [176].
Figure 7.
Diagram showing potential neuroprotective strategies to reduce tau aggregates. See text
for details.

Biomolecules 2016,6, 6 16 of 28
(1) MT-stabilizing agents, which as an approach does not address the accumulation of toxic
tau aggregates.
(2) Modulation of tau phosphorylation has been shown to prevent motor impairments in tau
transgenic mice [
173
]. Green coffee, a non-toxic small molecule, found to be an inhibitor of protein
phosphatase 2A methylesterase, was shown to improve cognitive and motor performance in mouse
models with tau pathology [174].
(3) A different approach, which does not depend on the phosphorylation theory, is based on
selective inhibition of pathological tau aggregation [
157
]. The problem in identifying suitable tau
aggregation inhibitors (TAI) is that most assays for tau aggregation are based on fibril formation (which
require relatively high concentrations of tau, tau constructs limited to the MT binding region with the
motif necessary for fibril formation, and a facilitator of aggregation, such as heparin). If compounds are
selected that dissociate preformed large aggregates into smaller (still toxic) oligomers then they may be
detrimental. In these assays, fibril formation is measured by a shift in fluorescence of an intercalating
reporter dye binding to β-sheet structures within tau fibrils.
(4) A more promising approach may be to target tau oligomers, whether these are intracellular or
extracellular. Purified tau oligomer species have been demonstrated to be neurotoxic (they directly
impair synaptic function and long-term potentiation, LTP)
in vitro
in a dose-dependent manner.
Extracellular tau levels measured in AD are more than four orders of magnitude lower than intracellular
tau concentrations, and as such may represent a more amenable pharmacological target.
(5) A quite different strategy is to target tau clearance—e.g., by rapamycin that induces
macroautophagy [
175
], inhibitors of Hsp90 chaperone protein that binds to misfolded proteins or by
immunotherapeutic approaches [176].
(6) Finally, it may be possible to target tau proteolysis directly. Cellular enzymes implicated
in tau proteolysis include caspases, calpains, cathepsins and a thrombin-like protease. Regardless
of the protease, it is reasonable to presume an irreversible loss of normal function of tau once it is
truncated. Disulfide-linked oligomers of tau can be observed in AD brains and cerebrospinal fluid (CSF)
samples, and show significant fragmentation, making them great potential targets for early diagnosis
of AD [
177
]. The major advantage of targeting tau proteolysis is that it may be more straightforward
to inhibit an enzymatic mechanism than aggregation, provided the proteolytic activity in question
can be shown to be rate-critical. It is possible that inhibition of truncation could prevent formation of
aggregation-prone fragments and also trans-synaptic/exosomal spread of tau pathology.
Recently, two drugs that targeted tau phosphorylation failed in phase 2 clinical trials [
178
]. This led
Wischik and collaborators [
157
] to propose that it is not abnormal tau phosphorylation that ought to be
reduced by drugs, but tau aggregation. The tau aggregation inhibitor LMTX (leucomethylthioninium
with a suitable counter-ion, Figure 8) is currently in three parallel Phase III clinical trials, with the first
outcomes expected in 2016 [
179
,
180
]. An older form of the molecule (methylthioninium chloride, MTC)
was found to have efficacy in mild/moderate AD in a Phase II clinical trial, in which 90% retardation
of disease progression could be demonstrated over 12 months [
180
]. These investigators stressed that
hyperphosphorylation of tau may not play a critical role in aggregation of tau and formation of PHF,
and that it may even have an inhibitory effect on tau-tau binding. Thus, it might be more important to
clarify proteolysis of tau protein (potentially at position Glu391, although this site may simply report
the C-terminal extent of the pathological binding domain) that enables the release of the C-terminal
fragment. This fragment is the one that appears to be important in the formation or propagation of
proteolytically stable tau oligomers that can spread to neighboring neurons trans-synaptically, further
propagate tau pathology and lead ultimately to formation of PHF. Recently, it has been proposed that
tau protein acetylation may be responsible for tau aggregation in AD. Grinberg and collaborators
detected tau acetylation at Lys274 in all tauopathies (both primary and secondary), except in AgD [
181
].
They hypothesized that tau acetylation could also promote the spreading of tau pathology (whereas in
AgD it could have a protective role in this respect).

Biomolecules 2016,6, 6 17 of 28
Biomolecules 2016, 6, 6 19 of 35
(6) Finally, it may be possible to target tau proteolysis directly. Cellular enzymes implicated in tau
proteolysis include caspases, calpains, cathepsins and a thrombin-like protease. Regardless of the
protease, it is reasonable to presume an irreversible loss of normal function of tau once it is truncated.
Disulfide-linked oligomers of tau can be observed in AD brains and cerebrospinal fluid (CSF) samples,
and show significant fragmentation, making them great potential targets for early diagnosis of AD [177].
The major advantage of targeting tau proteolysis is that it may be more straightforward to inhibit an
enzymatic mechanism than aggregation, provided the proteolytic activity in question can be shown to
be rate-critical. It is possible that inhibition of truncation could prevent formation of aggregation-prone
fragments and also trans-synaptic/exosomal spread of tau pathology.
Recently, two drugs that targeted tau phosphorylation failed in phase 2 clinical trials [178]. This led
Wischik and collaborators [157] to propose that it is not abnormal tau phosphorylation that ought to be
reduced by drugs, but tau aggregation. The tau aggregation inhibitor LMTX (leucomethylthioninium with
a suitable counter-ion, Figure 8) is currently in three parallel Phase III clinical trials, with the first
outcomes expected in 2016 [179,180]. An older form of the molecule (methylthioninium chloride,
MTC) was found to have efficacy in mild/moderate AD in a Phase II clinical trial, in which 90%
retardation of disease progression could be demonstrated over 12 months [180]. These investigators
stressed that hyperphosphorylation of tau may not play a critical role in aggregation of tau and
formation of PHF, and that it may even have an inhibitory effect on tau-tau binding. Thus, it might be
more important to clarify proteolysis of tau protein (potentially at position Glu391, although this site
may simply report the C-terminal extent of the pathological binding domain) that enables the release of
the C-terminal fragment. This fragment is the one that appears to be important in the formation or
propagation of proteolytically stable tau oligomers that can spread to neighboring neurons trans-
synaptically, further propagate tau pathology and lead ultimately to formation of PHF. Recently, it has
been proposed that tau protein acetylation may be responsible for tau aggregation in AD. Grinberg and
collaborators detected tau acetylation at Lys274 in all tauopathies (both primary and secondary),
except in AgD [181]. They hypothesized that tau acetylation could also promote the spreading of tau
pathology (whereas in AgD it could have a protective role in this respect).
Figure 8. Diagram of tau aggregation inhibitor LMTX (leucomethylthioninium with a
suitable counterion), and its presumed mode of action (inhibition of tau aggregation).
Figure 8.
Diagram of tau aggregation inhibitor LMTX (leucomethylthioninium with a suitable
counterion), and its presumed mode of action (inhibition of tau aggregation).
The investigators are currently putting most of their efforts into basic, preclinical and clinical
testing of methylene blue (MB) and its derivatives. MB is a phenothiazine that crosses the blood brain
barrier and acts as a redox cycler. Moreover, besides its beneficial properties as being able to improve
energy metabolism and to act as an antioxidant, it is also able to reduce tau protein aggregation. How
exactly LMTX and MTC exert their neuroprotective effects
in vivo
is not fully understood. MB (as MTC)
is able to reduce the amount of sarkosyl-insoluble tau in Drosophila that express human wild-type
tau [
182
], to disaggregate PHF isolated from AD brain [
152
] and to block prion-like processing of tau
protein in cell models [
153
]. Both MTC and LMTX have been shown to reduce tau pathology and
reverse behavioural deficits in transgenic mouse models of established pathology based either on the
repeat domain fragment or on full-length mutant tau P301S [
183
]. MB, together with its derivatives
(metabolites), azure A and azure B, is able to stimulate protein degradation and inhibit oxidative
damage [
184
] and also inhibit the activity of caspase-1 and caspase-3 [
185
]. MB given prior to the
onset of tau aggregation was also able to prevent learning and memory deficits in tau transgenic
mice [
186
], suggesting a potential preventative utility. Other possible inhibitors of tau aggregation are
rhodanine-based inhibitors, phenylthiazolyl-hydrazide inhibitors, N-phenylamines, phenothiazines
and benzothiazoles, and polyphenols and anthraquinones [187].
3. Conclusions
Although the pathogenic nature of the each type of protein deposit has been a controversial issue
for many years, it is now increasingly accepted that abnormal forms of tau protein are directly involved
in the initiation of neurodegerative processes. This conclusion is based primarily on the discovery
that dominant missense mutations in the MAPT gene are associated with dominant, familial forms of
FTD. Known polymorphisms in MAPT which confer susceptibility not only for AD and FTD, but other
neurodegenerative diseases as well, together with a possible additional novel disease locus near the
MAPT gene [
188
], strongly support the key role of tau protein not only in primary tauopathies but
also in the pathogenesis of LOAD and other secondary tauopathies.
Why disease onset takes decades before symptoms occur remains unclear at present, but current
results suggest a reduced ability to clear out misfolded, oligomerized and aggregated tau proteins
that increase with advancing age. As many drug discovery attempts based on the amyloid cascade
hypothesis have proved unsuccessful, and due to advances in our understanding of the role for tau in
AD pathogenesis [
189
], it is safe to conclude that tau protein will become an increasingly important

Biomolecules 2016,6, 6 18 of 28
therapeutic target for the future. The results of clinical trials with LMTX are eagerly awaited to confirm
whether a treatment for tauopathies is viable.
Acknowledgments:
This work was supported by The Croatian Science Foundation grant No. IP-2014-09-9730
(“Tau protein hyperphosphorylation, aggregation, and trans-synaptic transfer in Alzheimer’s disease:
cerebrospinal fluid analysis and assessment of potential neuroprotective compounds”) and European Cooperation
in Science and Technology (COST) Action CM1103 (“Stucture-based drug design for diagnosis and treatment
of neurological diseases: dissecting and modulating complex function in the monoaminergic systems of the
brain”). PRH is supported in part by NIH grant P50 AG005138. We also thank Mate Babi´c for help in preparation
of schematics.
Author Contributions:
Goran Šimi´c, Mirjana Babi´c Leko, Giuseppe Di Giovanni, and Patrick R. Hof conceived
the review. All authors contributed to drafting the work and revising it critically for important intellectual content.
Conflicts of Interest:
Charles Harrington (Chief Scientific Officer) and Claude Wischik (Executive Chairman) are
officers in TauRx Therapeutics Ltd and both are co-inventors on various patents related to tau protein.
Abbreviations
3R tau tau isoforms with three microtubule-binding repeats
4R tau tau isoforms with four microtubule-binding repeats
A adenine
Aβamyloid βprotein
AD Alzheimer’s disease
AD2 antibody specific for phospho-tau epitope Ser396
ADAM10 a disintegrin and metalloprotease domain 10
AgD argyrophilic grain disease
AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
APH-1 anterior pharynx-defective 1
APOE apolipoprotein E
APP amyloid precursor protein
AT8 antibody specific for phospho-tau epitopes Ser202 and Thr205
BACE β-site APP cleaving enzyme
BF Bayes factor
CBD corticobasal degeneration
CSF cerebrospinal fluid
DDPAC disinhibition-dementia-parkinsonism-amyotrophy complex
DM1 myotonic dystrophy type I
EOAD early-onset AD
fAD familial AD
FTD frontotemporal dementia
FTDP-17 frontotemporal dementia and parkinsonism linked to chromosome 17
FTLD frontotemporal lobar degneration
G guanine
Grb2 growth factor receptor-bound protein 2
HCHWA-D hereditary cerebral hemorrhage with amyloidosis-Dutch type
HDAC6 histone deacetylase 6
LMTX leucomethylthioninium
LOAD late-onset AD
LTP long-term potentiation
MAP2 microtubule-associated protein 2
MAPT microtubule-associated protein tau
MB Methylene blue
MCI mild cognitive impairment

Biomolecules 2016,6, 6 19 of 28
MN423 antibody for PHF core
MSTD multiple system tauopathy with presenile dementia
MT microtubule
NFT neurofibrillary tangles
NMDAR N-methyl-D-aspartate receptors
NT neuropil threads
O-GlcNAc O-linked N-acetylglucosamine
PD Parkinson’s disease
PEN-2 presenilin enhancer 2
PHF paired helical filaments
PSA puromycin-sensitive aminopeptidase
PSEN presenilin
PSEN1 presenilin 1
PSEN2 presenilin 2
PSP progressive supranuclear palsy
sAD sporadic AD
SDS sodium dodecyl sulfate
SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
SF straight filaments
SP senile plaques
TACE tumor necrosis factor alpha converting enzyme
Tau tubulin-associated unit
TNF-αtumor necrosis factor alpha
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