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Journal of Alzheimer’s Disease 27 (2011) 143–153
DOI 10.3233/JAD-2011-110623
IOS Press
143
Fulvic Acid Inhibits Aggregation and
Promotes Disassembly of Tau Fibrils
Associated with Alzheimer’s Disease
Alberto Cornejoa, Jos´
e M. Jim´
eneza,b, Leonardo Caballeroc, Francisco Melocand
Ricardo B. Maccionia,b,∗
aInternational Center for Biomedicine (ICC), University of Chile, ˜
Nu˜noa, Santiago, Chile
bLaboratory of Cellular and Molecular Neurosciences, Faculty of Sciences, University of Chile, ˜
Nu˜noa,
Santiago, Chile
cPhysics Department, University of Santiago, Santiago, Chile
Accepted 26 May 2011
Abstract. Alzheimer’s disease is a neurodegenerative disorder involving extracellular plaques (amyloid-) and intracellular
tangles of tau protein. Recently, tangle formation has been identified as a major event involved in the neurodegenerative process,
due to the conversion of either soluble peptides or oligomers into insoluble filaments. At present, the current therapeutic strategies
are aimed at natural phytocomplexes and polyphenolics compounds able to either inhibit the formation of tau filaments or
disaggregate them. However, only a few polyphenolic molecules have emerged to prevent tau aggregation, and natural drugs
targeting tau have not been approved yet. Fulvic acid, a humic substance, has several nutraceutical properties with potential
activity to protect cognitive impairment. In this work we provide evidence to show that the aggregation process of tau protein,
forming paired helical filaments (PHFs) in vitro, is inhibited by fulvic acid affecting the length of fibrils and their morphology.
In addition, we investigated whether fulvic acid is capable of disassembling preformed PHFs. We show that the fulvic acid is
an active compound against preformed fibrils affecting the whole structure by diminishing length of PHFs and probably acting
at the hydrophobic level, as we observed by atomic force techniques. Thus, fulvic acid is likely to provide new insights in the
development of potential treatments for Alzheimer’s disease using natural products.
Keywords: Alzheimer’s disease, atomic force microscopy, disassembly, fulvic acid, tau aggregation
INTRODUCTION
Alzheimer’s disease (AD) is the most common
type of dementia that is characterized by the for-
mation of two main protein aggregates in the brain:
senile plaques consisting of amyloid-(A) and
neurofibrillary tangles, which are composed mainly
∗Correspondence to: Dr. R.B. Maccioni, International Center for
Biomedicine, Avda, Vitacura 3568. D513, Vitacura,Santiago, Chile.
E-mail: rmaccion@manquehue.net.
of the microtubule-associated protein tau [1]. Tau
accumulates in a hyperphosphorylated state forming
intracellular deposits in AD called paired helical fil-
aments (PHFs) [2]. Physiologically tau stabilizes the
microtubule structure, but in the neurons of patients
with AD the microtubule system is believed to be dis-
rupted, with concomitant axonal transport deficits and
degeneration [3].
Recently, several data have shown that tau aggrega-
tion is the main event involved in the neurodegenerative
process. This process in AD correlates with the clinical
ISSN 1387-2877/11/$27.50 © 2011 – IOS Press and the authors. All rights reserved
144 A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD
progression of the disease and cognitive impairment
[4, 5]. The identification of mutations in the tau gene
in hereditary frontotemporal dementia has established
that dysfunction of tau protein is central to the neurode-
generative process [6]. Interestingly, improvement in
the cognition of a transgenic model displaying both
neurofibrillary tangles and senile plaques needs the
reduction of soluble tau, but not of senile plaques of
A[7]. Moreover, evidence for cytotoxicity of intra-
cellular aggregates comes from cellular models, where
overexpression of tau is deleterious to the neuron [8].
In the search of new molecules for the treatment of
AD, many drugs that have focused on Aaggrega-
tion have failed to stop the progression of the disease.
Immunization against Awas effective in reducing
amyloid plaque load, but it had little effect on improv-
ing cognitive function in patients [9, 10]. Also, a recent
study shows the failure of a Phase III clinical trial with
a␥-secretase inhibitor [11]. In this context, it seems
timely to consider alternative drug discovery strate-
gies for AD based on approaches directed at reducing
misfolded tau and compensating for the loss of normal
tau function [12]. Therefore, the development of small
molecules that inhibit the aggregation of tau appears
to be a valid therapeutic target for treatment of AD
and other tauopathies [13]. This hypothesis has been
favored by current findings on the compound methylth-
ioninium chloride (MTC, also known as methylene
blue), a previously described inhibitor of the aggre-
gation of tau of the phenothiazine’s family [14, 15]. A
recent study with this compound in Phase II clinical
trial shows an 81% reduction of cognitive decline with
the use of the compound compared to placebo [16].
Compounds described for their anti-aggregating
capacity in the formation of amyloid aggregates are
the polyphenols [17, 18]. In this context, synthetic
polyphenols have proved effective in the inhibition of
heparin-induced tau aggregation [15]. Following this
approach, current therapeutic strategies are aimed at
natural phytochemicals and polyphenolic extracts that
can either inhibit or disaggregate tau filament forma-
tion. It has been suggested that naturally occurring
phytochemicals have the potential to prevent AD based
on their anti-amyloidogenic, anti-oxidative, and anti-
inflammatory properties [19]. Despite this, there are
few phytocomplexes emerging to prevent tau aggre-
gation. Only a cinnamon extract and a grape seed
polyphenolic extract have been described for this pur-
pose [20, 21].
Fulvic acid is one of the most interesting phyto-
complex molecules. This is a mixture of polyphenolic
acid compounds resulting from the long-term micro-
bial degradation of lignin, among other sources [22].
It has several nutraceutical properties and is one of
the most interesting naturally-occurring phytochemi-
cals with its extremely high antioxidant properties and
apparent neuroprotective effect [23, 24]. For instance,
the interaction of prion protein with fulvic acid and its
inhibitory effect on the content of -sheet structure and
the formation of protein aggregates has been described
in detail [25].
Here, we investigated the effects of fulvic acid on
heparin-induced tau aggregation in vitro. The main
objective is the assessment of the inhibition of tau
aggregation in presence of fulvic acid by means of
three complementary techniques, namely thioflavin T
fluorescence (ThT) analysis of aggregation, atomic
force microscopy (AFM), and electron microscopy
(EM). The observation of formed aggregates by AFM
and EM allow us to conclude that fulvic acid inhibits
heparin-induced tau aggregation in vitro. On the other
hand, fulvic acid promotes the disassembling of tau
preformed fibrils. Thus, fulvic acid could provide a
new insight for developing treatments based on natural
products for AD.
MATERIAL AND METHODS
Materials
Fulvic acid standard (Suwanne River I 1S101F)
was obtained from the International Humic Substances
Society (IHSS, USA). This standard was extracted
from water of the Suwanne River in the Okefeno-
kee Swamp in south Georgia. Several structures has
been proposed by Leenheer and the third model is
shown in Fig. 1B [22]. Heparin was purchased from
Calbiochem and methylene blue was purchased from
Sigma-Aldrich.
Protein expression and purification
The sequence of htau 40 (longest isoform) was
donated gently by Dr. Eckard Mandelkow (Hamburg,
Germany). This fragment was cloned into pET-28a
vector (Novagen) to produce a His-tagged protein
and htau 40 was expressed in Escherichia coli strain
BL21(DE3). LB medium containing kanamycin was
inoculated with a stationary overnight culture. The
culture was grown at 37◦CtoOD
600 of 0.5–0.6 and
protein expression was induced by addition of 1 mM
IPTG for 4 h. The cells were pelleted and sonicated.
Recombinant tau was purified via a succession of
A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD 145
Fig. 1. Inhibitory effect of fulvic acid over PHF formation as mon-
itored by Thioflavin assay (ThT). A) Aggregation of tau fragment
4RMBD in the presence of fulvic acid at different concentrations
ranging between 0.1 to 200 M. The degree of inhibitory effect was
monitored by ThT and was plotted and represented as percentage of
residual aggregation as a function of fulvic acid concentration. The
IC50 value for inhibitory effect of Fulvic Acid was 37M. B) Sug-
gested model for the fulvic acid structure from the Suwanne River
standard [22].
Ni-Sepharose chromatography (equilibrated in 20 mM
NaH2PO4, 500 mM NaCl, and 20 mM imidazole, pH
7.4, elution with buffer 200 mM imidazole) and side
exclusion chromatography coupled to HPLC in a Pro-
teema 100 column (PSS, Germany) with buffer 50mM
NaH2PO4, 300 mM NaCl, pH 6.5. The purity of the
protein was verified on a Coomassie Brilliant Blue-
stained SDS-polyacrylamide gel. The protein was
concentrated and stored at –20◦C until use. The con-
centration of purified tau was determined using the
extinction coefficient at 280 nm (7700 M−1cm−1).
Tau fragment 4RMBD (htau244−372) was amplified
by using the plasmid for htau 40 as a template. The
PCR amplified sequence was subcloned into pET-28a
vector (Novagen) to produce a His-tagged protein.
The recombinant fragment 4RMBD was expressed in
Escherichia coli strain BL21 (DE3) and purified as
described above, via a succession of Ni-Sepharose
chromatography and size exclusion chromatography
coupled to HPLC. The concentration of purified
4RMBD was determined using the extinction coeffi-
cient at 280 nm (1520 M−1cm−1).
ThT fluorescence assay
The ThT fluorescence assay adopted here was mod-
ified from the reported by Pickhardt et al. [26] and
Crowe et al. [27]. Briefly, to examine the inhibition of
tau aggregation, the total volume of the reaction mix-
ture was 100 l, which included 20 M 4RMBD, 5 M
heparin in 100 mM sodium acetate, pH 7.0 with the ful-
vic acid standard at different concentrations. After 20 h
of incubation at 37◦C, addition of 100 lofa25M
solution of ThT was made and incubation continued for
1 h at room temperature prior to fluorescence reading.
Then, the fluorescence was measured in a Biotek Sin-
ergy 2 spectrofluorimeter (Bioteck Instruments, USA)
with an excitation wavelength at 440nm and emission
wavelength of 508 nm in a 96-well plate. Each experi-
ment was made at least in triplicate and the background
fluorescence was subtracted when needed. To exam-
ine the disassembly of preformed PHFs-like of tau,
the same reaction mixture was allowed to aggregate
for 20 h and then, the pure fulvic acid was added at
different concentrations.
Tau aggregation in vitro for EM and AFM
Aggregation for EM experiments was induced by
incubating htau 40 typically in the range of 45 M
in volumes of 100 Lat37
◦C in 10 mM Hepes,
100 mM NaCl, pH 7.4 buffer with the anionic cofac-
tor heparin (molar ratio of tau to heparin = 4:1) for
incubation time of 7 days with continuous shaking. Ful-
vic acid standard was resuspended in working buffer
and applied to the reaction at 200 M final concen-
tration prior to heparin addition. 4RMBD and htau 40
aggregation experiments for AFM were induced sep-
arately. 4RMBD aggregation was incubated at 20 M
of protein and 5 M of heparin with 20 mM Hepes
pH 7.4, 25 mM NaCl for incubation time of 24 h
with continuous shaking. Fulvic acid standard was
resuspended in working buffer and applied to the reac-
tion at 120 M final concentration prior to heparin
addition. Negative control was developed with only
4RMBD (without both heparin and Fulvic acid) and
another with methylene blue at 100 M. The inhi-
bition experiments with htau 40 were developed as
146 A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD
described above. For disassemble experiments, full
length tau was induced for 6 days and then ful-
vic acid was added at 200 M for the same time.
The preformed fibril control was induced for 12
days.
Transmission electron microscopy
To prepare the samples for electron microscope
observation, 5 L of protein solutions and 5 Lof
buffer (10 mM Tris-HCl, pH 7.4 KCl 50 mM) were
placed on formvar/carbon coated copper grids and
were left at room temperature for 5 min. Then, the
samples were negatively stained with 2% (w/v) uranyl
acetate for 15 s. All samples were examined with the
help of a JOEL EM1200 electron microscopy at 80 kV.
Atomic force microscopy
Tau protein was diluted in 10 mM Tris-HCl, pH 7.4,
50 mM KCl and then immobilized onto highly ordered
pyrolitic graphite (HOPG) to a final concentration of
5M. A drop of tau protein solution was deposited
onto HOPG surface (30 l) and adsorbed for 30 min.
The excess of protein was removed by washing with
abundant 10 mM Tris-HCl, pH 7.4, 50 mM KCl solu-
tion. In order to avoid spontaneous disassembly all
atomic force images were obtained in liquid environ-
ment using 10 mM Tris-HCl, pH 7.4, 300 mM KCl.
AFM imaging was performed in tapping (intermit-
tent contact) mode in fluid using a Nanoscope III
(Veeco, CA) and Si3N4cantilevers (NPS series, Veeco)
exhibiting spring constants of 40–60 N/m at resonance
frequencies in buffer of 6 to 10 kHz [28]. To achieve
minimal imaging forces between AFM stylus and sam-
ple, the drive amplitude was set between 0.5 and 1.0V,
and the amplitude set point was adjusted manually to
compensate for the thermal drift of the AFM. The force
applied by the scanning process of AFM ranges from
80 to 100 pN.
Data analysis
The ThT fluorescence data were adjusted to a sig-
moidal model and graphed with Origin 6.0 software.
Fibril lengths were obtained with the program of
Nanoscope III system from the AFM images and
these data were analyzed with homemade software
(developed at the University of Santiago) after being
normalized.
RESULTS
Fulvic acid inhibits the 4RMBD tau fragment
aggregation process
For screening the inhibition of tau protein aggrega-
tion, the fourth microtubule binding domain (4RMBD)
was chosen instead of full length tau protein. Indeed,
it is well known that 4RMBD repeat is more prone to
aggregation and assembles into PHFs, the hallmark of
AD. In order to test the capacity of fulvic acid (Fig. 1B)
to inhibit aggregation we used three methods including
fluorescence spectroscopy, AFM, and EM. Figure 1A
shows the inhibition isotherm at different concentra-
tions of fulvic acid, as monitored by ThT, a compound
that is able to bind to fibrils containing -sheet struc-
ture promoting changes in the fluorescence spectrum
[29]. Fulvic acid in a concentration of 120 Mwas
able to inhibit in 85% the process of aggregation indi-
cating that structures diminish when fulvic acid is
incubated with 4RMBD fragment. In order to check
the correct formation of fibrils, we first induced the
aggregation process, observing under AFM the sev-
eral structures on the HOPG surface. These structures
resemble oligomers and longest fibrils (Fig. 2A, Con-
trol). After treatment of fibrils with fulvic acid, only
oligomeric structures were observed over the surface
of HOPG (Fig. 2B). A bigger magnification, Fig. 2b
inset, shows unstructured oligomers. The experiment
was repeated ten times and twenty images took ran-
domly were analyzed for each run. Fibrils formation
was never observed on HOPG surface in the presence
of fulvic acid, suggesting that the aggregation pro-
cess was strongly inhibited. This fact indicates that
fulvic acid could be interacting with different regions
of 4RMBD avoiding almost completely the process
of aggregation. Also a negative control is shown in
Fig. 2C suggesting that 4RMBD in absence of heparin
does not form either oligomers or fibrils. On the other
hand, it is well documented that methylene blue has a
strong inhibitory properties on tau aggregation process
[15]. In this context we were interested on investigat-
ing whether methylene blue is still able to inhibit the
4RMBD at low concentration (IC50) and to exhibit the
same pattern on HOPG surface as fulvic acid does.
Interestingly, the analysis of AFM images of methy-
lene blue (Fig. 2D) shows oligomers and few fibrils on
HOPG surface, suggesting that despite the high degree
of inhibition of aggregation exhibited by methylene
blue in ThT fluorescence assay [15], this inhibition
does not avoid the filaments formation.
A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD 147
AB
CD
Fig. 2. The assembly of 4RMBD tau fragment is inhibited by fulvic acid. A) The aggregation of 4RMBD was induced by heparin and analyzed
by atomic force microscopy over a HOPG surface (Control). Bar =1 m. B) The structures formed in presence of 4RMBD as analyzed by atomic
force microscopy after treatment with 120 M fulvic acid. Bar= 1 m. Inset b) Detailed image resulting from atomic force microscopy of fibrils
treated with fulvic acid. Bar =500 nm. C) Negative control 4RMBD in absence of both heparin and fulvic acid. Bar = 1 m. D) Internal control
of the inhibition process using methylene blue at 100 Mm (instead of fulvic acid) as analyzed by atomic force microscopy. Bar = 500nm.
Fulvic acid is able to inhibit the aggregation
process of full length tau protein
Even though our previous experiments have shown
that fulvic acid is able to inhibit the aggregation process
and to avoid the fibrils formation, it was pertinent to
investigate whether the fulvic is capable to inhibit the
aggregation process of full length tau protein as well.
To rule this out, we induced the aggregation of hTau
40 in the absence or presence of fulvic acid. As we
observed in Fig. 3A, the longest fibrils structures and
oligomers were formed after heparin induction. The
most abundant elements on HOPG surface were fibrils
whose lengths are varying between ∼1m and 3 m.
In turn, Fig. 3B shows that pre-fibril and oligomeric
structures are also well formed after heparin induc-
tion whose lengths range from 30 nm to 100 nm, in
the case of oligomers, and 300 nm in the case of pre-
fibril structure. After treatment of full length tau protein
with fulvic acid (Fig. 4A), the presence of fibrils on
HOPG surface was not detected over several trials
since the length of oligomeric structures or pre fibrils
were clearly affected. Figure 4B shows in detail the
oligomeric structures that remain after the aggregation
148 A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD
A
B
Fig. 3. Full length tau recombinant protein assembles into fibrils.
A) Fibril formation of hTau 40 protein was induced by heparin and
analyzed by atomic force microscopy. Several fibrils and oligomers
over HOPG surface are observed. Bar= 500 nm. B) Oligomers and
pre fibril structures are observed by atomic force microscopy after
heparin induction of hTau 40 tau protein. Arrow is indicating an
oligomeric and a pre-fibril structure. Bar =100 nm.
process induced by heparin in the presence of fulvic
acid. In order to investigate in more detail these obser-
vations, we used EM. EM images reveal the presence
of several fibrils after heparin induction (Fig. 5A) and
also the presence of typical PHF (Fig. 5C) as control
images. Otherwise, in the case of hTau 40 treated with
fulvic acid, we were not able to observe fibrils but
only a few elements resembling pre fibril structures
(Fig. 5B). Figure 5D shows in more detail a pre-fibril
structure, but we did not observe either straight fila-
ments or PHF as observed in control images (Fig. 5A
and Fig. 5C).
A
B
Fig. 4. Fulvic acid treatment blocks PHF-like fibrils of tau
molecules. A) Oligomeric structures rather than tau fibrils are
observed by atomic force microscopy in the presence of 200 M
fulvic acid. Bar = 500 nm. B) Detailed observation of few oligomeric
structures present after treatment with fulvic acid. Arrows indicate
an oligomeric structure. Bar = 100 nm.
On the other hand, in order to analyze differences
in fibril length of tau protein control and tau protein
treated with fulvic acid, we analyzed all these data
with our homemade software. First, the lengths of fib-
rils were obtained using Veeco software of Nanoscope
III and all data were normalized before analyses. Fig-
ure 6 shows the relative frequencies as a percentage of
tau control (fulvic acid absence) and tau-treated (with
fulvic acid). As seen in Fig. 6, the relative frequency of
length in tau protein treated with fulvic acid is about to
200 nm, suggesting that the aggregation process was
inhibited by fulvic acid, avoiding fibril formation. In
A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD 149
AB
CD
Fig. 5. Fibrils formation of heparin-induced hTau 40 and treated with fulvic acid as analyzed by electron microscopy. A) Control of fibrils of
full length tau protein induced by heparin. Bar = 500nm. 10,000 ×magnification. B) Fibril formation is clearly diminished and only a small
number of structures are observed in the presence of 200 M fulvic acid. Bar= 500 nm. 15,000 ×magnification. C) PHF-like structures formed
with heparin, and twisted structures suggest that fibers are two strands. Bar = 500nm. 10,000 ×magnification. D) A few pre fibrils were found in
heparin-induced filaments from full length tau protein after treatment with fulvic acid. The length and thickness is diminished in those filaments.
Bar = 500 nm. 15,000 ×magnification.
contrast, the lengths of tau protein control fibrils (red
bars) were more heterogeneous.
Fulvic acid promotes the disassembly of
preformed tau fibrils
So far, we demonstrated that fulvic acid is an
inhibitory compound of both 4RMBD and full length
tau protein. However, it is interesting to prove whether
fulvic acid is capable of disassembling preformed
fibrils of tau, whose structures are responsible for neu-
ronal cell death [26].
In this context, we first induced tau fibril formation
by heparin (as control) and then after this formation,
the filaments were assayed with fulvic acid (200 M).
Interestingly, using fluorescence spectroscopy, we
found that fulvic acid disassembles preformed fibril tau
in a dose-depending manner (Fig. 7). In addition, we
were able to determine the IC50 concentration of this
compound (95 M). After this analysis we focused on
demonstrating, by AFM, which fibril structures remain
after fulvic acid treatment. Figure 8A shows that sev-
Fig. 6. Data analysis of the filaments length of hTau 40 protein
treated with fulvic acid. Modal relative frequencies of the length
of control tau fibrils (red clear) and lengths of fibrils of tau treated
with fulvic acid (Blue darker). All data were normalized and ana-
lyzed by a homemade software for the fibers analysis produced at
the University of Santiago.
eral structures are formed after heparin induction of
full length tau protein. Heterogeneous populations
(Fig. 8B) were also observed. Analysis by Nanoscope
150 A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD
Fig. 7. Fulvic acid promotes disassembly of preformed fibrils of
tau fragment 4RMBD as monitored by fluorescence spectroscopy
(ThT). For the disassembly experiments, tau fragment 4RMBD was
induced to aggregate into PHF-like structures for 20 h as described,
and afterward the fibrils were exposed for another 20 h to fulvic acid.
The process of inhibition is represented as a percentage of aggre-
gation as a function of fulvic acid concentration. The disassembly
concentration for 50% inhibition, DC50 value, is 95 M.
software of fibrils allows us to determinate the height
and thickness of structures observing a regular dis-
tribution of fibrils whose heights vary between 8 to
10 nm. Thicknesses of fibrils are also regular, varying
between 50 to 100 nm (Fig. 8C). Typical dimensions
vary depending on which structure is analyzed but
clearly the structures are forming part of heterogeneous
population (Fig. 8A, B).
In addition, to determine which effects are observed
over tau fibrils formed after treatment with fulvic acid,
we analyzed this effect by AFM. Figure 9A shows a
marked effect of fulvic acid on preformed fibrils dimin-
ishing the number of structures and their lengths. In
Fig. 9B we observe a clear effect of fulvic acid on the
filamentous structures, with a phenomenon of short-
ening of these elements. Thickness of fibrils treated
with fulvic acid are very similar to controls but their
heights are diminished (Fig. 9C). To determine in inde-
pendent and normalized experiments whether fulvic
acid has a significant effect on fibril lengths, com-
pared with untreated controls, we used the homemade
software. Thus we were able to determine the relative
frequencies. Figure 10A shows that preformed fibrils
are effectively disassembled (blue bars) instead of con-
trols (red bars). The controls also show a heterogeneous
population of fibrils being distributed along the lengths
axis. Otherwise, the highest relative frequencies coin-
cide with fibrils having in length less than 200 nm,
showing that fulvic acid treatment of preformed fibrils
A
BC
Fig. 8. Characterization of the assembly process of full length tau as
monitored by atomic force microscopy. A) AF image of fibrils for-
mation induced by heparin and analyzed by atomic force microscopy
on HOPG surface. Bar = 1 m. B) Detailed image of fibrils for-
mation from hTau 40, co-existing also pre-fibrils and oligomeric
structures. Bar = 500nm. C) Analysis of the thickness and height (in
the 3D structural analysis) of fibrils carried out with a Nanoscope III
software. Red and blue represent two different fibers. Bar = 300 nm.
is a very effective compound against either aggregation
or disassembly process.
DISCUSSION
Neurodegenerative disorders such Alzheimer’s dis-
ease (AD) are characterized by misfolding and/or
aggregation processes affecting specific brain regions.
One interesting aspect of these misfolding and/or
aggregation processes is that it could be handled by
efficient processes of clearance such autophagy, prote-
olysis, and proteosome system. Instead, in aging and
disease the accelerated process of oligomerization and
subsequent fibril formation in general, can increase the
stress levels and also appears to reduce the ability of
cells to avoid the protein aggregate burden [30–32].
A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD 151
A
BC
Fig. 9. Fulvic acid induced disassembly of full length tau fibrils as
analyzed by atomic force microscopy. A) Images of filaments result-
ing from disassembly of tau aggregates by fulvic acid treatment
on HOPG surface. Bar = 1 m. B) Detailed image of disassem-
bly and shortening process of fibrils after fulvic acid treatment.
Bar = 500 nm. C) Analyses of thickness and height of fibrils after ful-
vic acid treatment, as analyzed with Nanoscope III software (Veeco,
digital Instrument Metrology group). Bar =300 nm.
The Acascade is placed upstream of tau, a notion
supported by an AD mouse model [32]. As such, it is
very interesting that preventing tau depletion in mice
can avoid Apathology [33].
Recently, it has been demonstrated that a peculiar
grape seed polyphenolic extract (GSPE) may attenuate
tau protein misfolding leading to form either aggre-
gates or fibrils, a critical step for the progression of
the disease [34]. In addition, interesting studies have
shown that vitamin A and carotene have inhibitory
properties that disassemble Apreformed fibrils [30].
Moreover, all-trans retinoic acid has strong antioxi-
dant properties able to reduce the burden of oxidative
stress associated with AD [31]. In the context of the
present work, humic substances such as fulvic acid
have shown some effects on protease K resistance
Fig. 10. Data analysis of the disassembly of hTau 40. Modal rela-
tive frequencies of the lengths of tau fibrils induced by heparin (red
clear) and preformed fibrils treated with fulvic acid (blue darker)
are represented as percentage of aggregation as a function of fulvic
acid concentration. All data were normalized and analyzed by our
home-made software for morphometric parameters of fibers.
and cell internalization of human prion protein [25].
Despite all these promising results, only a few com-
pounds are available to treat or prevent symptoms of
AD generated by the malfunction of tau protein. Thus,
the present work aimed to test the action of fulvic acid,
a molecule which has been extensively characterized
in previous studies [22]. We used aggregation model
4RMBD and full length tau protein to assess the poten-
tial role of fulvic acid in preventing aggregation and/
or disassembly of tau protein. First, we described anti
aggregation properties of fulvic acid against 4RMBD
by fluorescence spectroscopy suggesting that aggrega-
tion process is strongly inhibit and as well the -sheet
conformation suggesting that stability of -conformers
are diminished. Using AFM, we were able to show
that 4RMBD is diminished on HOPG surface and
that the structures remaining on HOPG surface resem-
bled unstructured oligomers. Interestingly, when we
decided to test methylene blue as an internal control
of our experiments by AFM, we were able to observe
the presence of oligomeric structures as well as some
fibrils, showing that a diminishing of ThT fluores-
cence intensity does not reflect an inhibitory effect of
methylene blue on tau filaments over HOPG surface.
Otherwise, although the IC50 of fulvic acid is a little
higher, we did not observe fibrils formation on AFM
assay suggesting that a different pathway of inhibition
is involved.
While our experiments have shown that tau aggre-
gation is inhibited by fulvic acid, it was pertinent to
find out whether the same effect could be observed on
full length tau protein. Interestingly our observations
152 A. Cornejo et al. / Fulvic Acid Inhibits Aggregation and Promotes Disassembly of Tau Fibrils with AD
reveal a marked effect on fibril formation given that
only oligomeric or pre fibril structures were seen. Con-
sistently, in control images typical PHFs were observed
by EM instead of full length tau protein treated with
fulvic acid. In addition, the lengths analyzed in control
and full length tau protein experiments treated with
fulvic acid revealed a marked difference. All data sug-
gest that fulvic acid effectively acts by affecting the
aggregation process of either 4RMBD or full length
tau protein.
On the other hand, it is well known that fibril for-
mation is a crucial event in the pathogenesis of AD.
Therefore, we were interested in observing which
effects could be exerting fulvic acid on preformed tau
filaments. Interestingly, we found that fulvic acid is
able to disassemble preformed fibrils of tau protein. It
is well known that forces of interaction between HOPG
and proteins involved hydrophobic interactions. In this
regard it is interesting to point out data arising from
AFM images of tau protein treated with fulvic acid,
where we observed a decrease in tau protein inter-
actions as compared with HOPG. This was observed
despite forces being applied between 80 to 100 pN, in
order to avoid disassembly of fibrils, a fact that might
suggest that hydrophobic interactions are diminished
by fulvic acid. The inhibitory effect of fulvic acid can
occur by association between fulvic and tau monomers
or oligomers, therefore avoiding the final fibril forma-
tion process. In this respect, it seems important that
fulvic acid is able to disassemble preformed fibrils
despite that several minor structures are seen on HOPG
resembling unstructured oligomers or pre-fibrils. How-
ever, it has been demonstrated with other polyphenols
that these structures can be part of other non-toxic path-
ways [35]. Finally we concluded that fulvic acid is a
promising molecule able to inhibit and disassembly
tau fibrils. Subsequent studies are necessary in order
to determinate its activity in vivo.
ACKNOWLEDGMENTS
This work was supported by grants 1080254,
1110373 and 1100603 from Fondecyt, grant 10-ANT-
8051 from CORFO, VRI project from Fondef and by
the Alzheimer´s Association, USA. We thank Carlos
Carrasco for valuable help and the International Center
for Biomedicine and the University of Chile for travel
fellowship to JMJ.
Authors’ disclosures available online (http://www.j-
alz.com/disclosures/view.php?id=897).
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