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Beneficial Effects of Sideritis scardica and Cichorium spinosum against Amyloidogenic Pathway and Tau Misprocessing in Alzheimer’s Disease Neuronal Cell Culture Models

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Background: Natural products are a significantly underutilized source of potential treatments against human disease. Alzheimer's disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by recent findings. Objective: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet. Methods/results: After the detailed characterization of the extracts' composition using LC-HRMS methods, they were evaluated on two AD neuronal cell culture models, namely the AβPP overexpressing SH-SY5Y-AβPP and the hyperphosphorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and subsequently their downstream effects on AβPP and tau processing pathways were investigated in the SH-SY5Y-AβPP and PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly decrease total tau, the activation of the GSK3β, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Furthermore, both extracts appear to promote AβPP processing through the alpha, non-amyloidogenic pathway, albeit through partly different mechanisms. Conclusions: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or treatment of AD, and merit further investigations at the in vivo level.
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Journal of Alzheimer’s Disease xx (20xx) x–xx
DOI 10.3233/JAD-170862
IOS Press
1
Beneficial Effects of Sideritis scardica
and Cichorium spinosum against
Amyloidogenic Pathway and Tau
Misprocessing in Alzheimer’s Disease
Neuronal Cell Culture Models
1
2
3
4
5
Ioanna Chalatsaa,2, Demetrios A. Arvanitisb,2, Eleni V. Mikropoulouc, Athina Giaginia,
Zeta Papadopoulou-Daifotid,1, Nektarios Aligiannisc, Maria Halabalakic, Anthony Tsarbopoulosd,e,
Leandros A. Skaltsouniscand Despina Sanoudoua,b,
6
7
8
a4th Department of Internal Medicine, Clinical Genomics and Pharmacogenomics Unit, Medical School,
National and Kapodistrian University of Athens, Athens, Greece
9
10
bMolecular Biology Division, Center for Basic Research, Foundation for Biomedical Research of the Academy
of Athens, Athens, Greece
11
12
cDepartment of Pharmacognosy and Natural Product Chemistry, Faculty of Pharmacy, National and Kapodis-
trian University of Athens, Athens, Greece
13
14
dDepartment of Pharmacology, Medical School, National and Kapodistrian University of Athens, Athens, Greece15
eDepartment of Bioanalytical, GAIA Research Center, The Goulandris Natural History Museum, Kifissia, Greece16
Handling Associate Editor: Antonios Politis17
Accepted 9 May 2018
Abstract.18
Background: Natural products are a significantly underutilized source of potential treatments against human disease.
Alzheimer’s disease (AD) is a prime example of conditions that could be amenable to such treatments as suggested by
recent findings.
19
20
21
Objective: Aiming to identify novel potentially therapeutic approaches against AD, we assessed the effects of Cichorium
spinosum and Sideritis scardica extracts, both distinct components of the Mediterranean diet.
22
23
Methods/Results: After the detailed characterization of the extracts’ composition using LC-HRMS methods, they were
evaluated on two AD neuronal cell culture models, namely the APP overexpressing SH-SY5Y-APP and the hyperphos-
phorylated tau expressing PC12-htau. Initially their effect on cell viability of SH-SY5Y and PC12 cells was examined, and
subsequently their downstream effects on APP and tau processing pathways were investigated in the SH-SY5Y-APP and
PC12-htau cells. We found that the S. scardica and C. spinosum extracts have similar effects on tau, as they both significantly
decrease total tau, the activation of the GSK3, ERK1 and/or ERK2 kinases of tau, as well as tau hyperphosphorylation. Fur-
thermore, both extracts appear to promote APP processing through the alpha, non-amyloidogenic pathway, albeit through
partly different mechanisms.
24
25
26
27
28
29
30
31
1This author passed away on March 17, 2016.
2These authors contributed equally to this work.
Correspondence to: Despina Sanoudou, PhD, FACMG, 4th
Department of Internal Medicine, Clinical Genomics and
Pharmacogenomics Unit, “Attikon” Hospital, Medical School,
National and Kapodistrian University of Athens, Rimini 1,
Chaidari 124-62, Greece. Tel.: +30 210 7462532; E-mail:
dsanoudou@med.uoa.gr.
ISSN 1387-2877/18/$35.00 © 2018 – IOS Press and the authors. All rights reserved
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Conclusions: These findings suggest that C. spinosum and S. scardica could have a notable potential in the prevention and/or
treatment of AD, and merit further investigations at the in vivo level.
32
33
Keywords: Alzheimer’s disease, amyloid, amyloidosis, Cichorium spinosum, Mediterranean diet, mountain tea, neurode-
generative diseases, prevention, Sideritis scardica, tauopathies
34
35
INTRODUCTION32
Alzheimer’s disease (AD) is a progressive neu-
33
rodegenerative disease, the most common form of
34
diagnosed dementia (>44 million AD patients world-35
wide), and the 6th leading cause of death in the USA.36
By 2050, more than 100 million people are expected37
to be affected worldwide [1]. Currently, there are
38
no effective treatments, while the FDA approved
39
compounds exhibit short-term benefits and can have40
serious side effects [2, 3]. Natural products with neu-41
roprotective activities are believed to hold significant
42
promise in preventing or treating AD [4–8].43
It is anticipated that some of these products could44
demonstrate beneficial effects by preventing key AD
45
pathogenetic mechanisms namely the amyloidogenic46
and neurofibrillary tangles (NFTs) pathways [9]. The47
amyloid pathway is implicated in neuronal activity48
and differentiation, including synapse formation and49
transmission [10]. Under physiological conditions,50
the amyloid-protein precursor (APP) is processed51
by the alpha or beta proteolytic pathways. Through
52
the non-amyloidogenic alpha pathway, APP is53
cleaved by the alpha [11] and gamma secretases to
54
produce soluble APP alpha (sAPP) and APP-
55
C83 [12–14]. The active gamma secretases, PSEN1
56
and PSEN2, are the products of the respective
57
full-length protein cleavage and amino-/carboxy-58
terminal fragment (NTF/CTF) heterodimerization59
[15, 16], and are also encountered in the form of60
multiprotein complexes [17, 18]. Through the amy-
61
loidogenic beta pathway, APP is cleaved by beta62
secretase (BACE1) releasing sAPPto the extra-63
cellular space and leaving APP-C99 in the plasma
64
membrane. APP-C99 can be subsequently pro-
65
cessed by gamma secretase into amyloid-(A)of
66
different lengths (ranging from 37 to 46 amino acids)
67
and APP intracellular C-terminal domain (AICD)68
[12, 19]. Some species of Aare particularly toxic69
causing synaptic failure and neuronal death [20].70
The intracellular fibrils and NFTs formed dur-71
ing AD development consist primarily of the72
hyperphosphorylated and misfolded microtubule-
73
associated protein, tau. Normally located at the
74
axons, tau is important for neuronal differentiation
75
and development, maintenance of cellular morphol- 76
ogy and polarity, as well as axonal transport of 77
organelles, vesicles, or molecules [21]. Tau is sub- 78
jected to extensive post-translational modifications, 79
predominantly phosphorylation in >85 sites [22]. 80
The aberrant phosphorylation of tau in AD leads 81
to its dissociation from microtubules, microtubule 82
destabilization, loss of dendritic microtubules 83
and synapses, interruption of axonal transport, 84
plasma membrane degeneration, and eventually 85
neuronal loss [23, 24]. The hyperphosphorylated 86
tau molecules tend to self-assemble intracellu- 87
larly into filaments forming NFTs. Following the 88
death of tangle-bearing cells, tau filaments are 89
released in the extracellular space as neurotoxic 90
“ghost” tangles stimulating activation of microglial 91
cells and progressive neuronal degeneration 92
[2, 25, 26]. 93
The identification of natural products that could 94
disrupt the amyloid cascade or tau misprocessing and 95
prevent the accumulation of amyloid plaques or NFTs 96
is an area of intense research in the battle against AD. 97
Since dietary habits seem to significantly affect the 98
prevalence of cognitive impairment in a population 99
[27, 28], it has been proposed that the regular intake 100
of certain classes of compounds, such as antioxidants, 101
might act protectively against neuronal cell oxidation 102
and cognitive decline [29, 30]. Natural phenols, such 103
as those encountered in fresh fruits, green vegeta- 104
bles, red wine, and olive oil, are often regarded as 105
health-promoting options for maintaining cognitive 106
health [31–33]. In that sense, the model that combines 107
all of these nutritional elements, the Mediterranean 108
diet, should be thoroughly examined as a potent pre- 109
ventive tool in the battle against neurodegenerative 110
diseases. 111
The Mediterranean diet constitutes an exemplary 112
model diet with its benefits varying from low rates 113
of heart disease to higher survival rates [34, 35]. In 114
recent years, heightened attention has been drawn 115
to the link between the Mediterranean diet and 116
mental function in older adults [36]. In particular, 117
individuals adhering to a Mediterranean style diet 118
seem to have a reduced risk for developing AD [37] 119
and mild cognitive impairment (MCI), as well as 120
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for deterioration from MCI to AD [38]. Recently,121
a population study in rural Crete, demonstrated a
122
lower prevalence for dementia compared to global123
data [39]. Consequently, the study of the Cretan /124
Mediterranean diet, might serve as a rich source of125
new bioactive natural products.
126
Among the key components of the Mediterranean127
diet, the decoction of S. scardica leaves, commonly128
known as “mountain tea”, represents a daily habit129
and a traditional remedy. As such, the beneficial
130
properties of plants of the genus Sideritis have been131
extensively studied. A recent study demonstrated that132
S. scardica extracts inhibited the uptake of the neuro-
133
transmitters serotonin, noradrenaline, and dopamine,134
which are involved in multiple neurological disorders
135
[40], while other species of the Sideritis genus have136
been shown to exert antioxidant and anxiolytic-like137
properties [41], anticholinesterase activity [42, 43],
138
and to improve the spatial learning and memory in139
mice with A-induced amnesia [44]. Sideritis spp.140
extracts seem to reduce the amyloid plaque burden141
in transgenic mice, and significantly ameliorate their142
memory function [45], while a dietary supplement of143
S. scardica and selected B vitamins has been found144
to reduce stress-induced impairment of mental func-
145
tion in young adults [46]. S. scardica extract has also
146
been found to improve cognition and mental func-147
tion by modulating the AMPA receptor dependent
148
neurotransmission involved in synaptic plasticity and149
age-related cognitive decline [47].150
Another highly valued and extensively consumed
151
ingredient of the Mediterranean diet and especially152
the Cretan diet is the wild edible greens (ch´
orta)
153
of Crete, C. spinosum, an endemic Mediterranean
154
plant also known as “stamnagkathi” [48, 49], and it155
is thought to improve liver function [50]. Recently156
the C. intybus L. member of the Cichorium genus157
was suggested to improve amnesia and memory158
process impairment in rats [51]. Importantly, mul-159
tiple compounds present in C. spinosum extracts,
160
such as aesculetin, cichoric acid, chlorogenic acid,161
3,4-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid,
162
4,5-dicaffeoylquinic acid, have been demonstrated to163
play a neuroprotective role [52–56]. Furthermore, the164
C.spinosum compound quercetin-3-O-glucuronide165
significantly reduces the generation of Apeptides166
and improves AD-type deficits in hippocampal for-167
mation basal synaptic transmission and long-term168
potentiation [57]. Furthermore, both S. scardica and
169
C. spinosum contain high levels of phenolic sub-
170
stances, such as phenolic acids and flavonoids, and
171
they have exhibited potent antioxidant activities in
172
in vitro models [58–61]. They therefore merit fur- 173
ther investigation for their potential in modulating 174
the molecular milieu of AD. 175
In this context, we assessed the effects of these two 176
plants’ natural extracts, distinct components of the 177
Mediterranean diet, in two established in vitro (cell 178
line) models of AD. We demonstrate that treatment 179
with “mountain tea” (S. scardica) or “stamnagkathi” 180
(C. spinosum) extracts it modulates multiple steps of 181
the APP misprocessing and tau hyperphosphoryla- 182
tion pathways, suggesting a potential preventive and 183
possibly therapeutic potential in AD. 184
MATERIALS AND METHODS 185
Plant material extraction 186
For S. scardica, the dry plant material was 187
extracted with methanol using ultrasounds for 2 h, at 188
room temperature and the extract was concentrated 189
to dryness after filtration. For C. spinosum, the fresh 190
stems and leaves were boiled with distilled water in 191
a ratio of 1 kg plant material / 2 L of water and the 192
obtained decoction was filtered and lyophilized. 193
UHPLC-ESI(-)-HRMS analysis 194
Liquid chromatography analysis for S. scardica 195
was performed on an Accela®High-Speed LC Sys- 196
tem (Thermo Scientific) and for C. spinosum on an 197
Acquity®UPLC System (Waters). For both extracts, 198
detection was carried out on a LTQ-Orbitrap®199
XL hybrid mass spectrometer equipped with an 200
ESI source (Thermo Scientific), in negative mode. 201
Due to the different polarity of the extracts’ con- 202
stituents, two different gradient separation methods 203
were used. For S. scardica qualitative analyses, sep- 204
aration was achieved on a Fortis®C18 column 205
(100 mm ×2.1 mm, 1.7 m) using a gradient of water 206
containing 0.1% (v/v) formic acid (A) and acetonitrile 207
(B). Elution started with 95% A for 3 min and 208
decreased to 0% A in 21 min. These conditions 209
were maintained for 2 min before reverting to the 210
initial conditions for 7-min of re-equilibration. For 211
C. spinosum analysis, separation was achieved on 212
a Fortis®C18 column (150 mm ×2.1 mm, 1.7 m) 213
using the same solvent system. Elution started with 214
95% A and decreased to 5% in 23 min. These con- 215
ditions were maintained for 3 min before reverting 216
to initial conditions in 2 min, for a final 3-min 217
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re-equilibration. In both cases, the column was main-218
tained at 40C and the flow rate was set at 0.4 mL/min.219
10 L of water extracts at 200 g/mL were injected.220
MS data were acquired in negative-ion mode, in the221
full scan of 113–1000 m/z, with a resolution of 30000.222
Capillary temperature was set at 350C, whereas223
source voltage was 2.7 kV in ESI-. Tube lens and224
capillary voltage were tuned at –40 V and –10 V,225
respectively. Finally, nitrogen was used as sheath gas226
(40 arbitrary units) and auxiliary gas (10 arbitrary
227
units). For both extracts, a sample was prepared for228
analysis at a concentration of 0.2 mg/mL in a mixture229
of the mobile phase.
230
Cell culture and differentiation231
The human neuroblastoma SH-SY5Y and rat232
pheochromocytoma PC12 cell lines were used as well
233
established neuronal models that can differentiate
234
into neuron-like cells. Furthermore, SH-SY5Y-APP235
cells inducibly over-expressing APP695 (a kind236
gift of Dr S. Efthimiopoulos, Faculty of Biology,
237
National and Kapodistrian University of Athens,238
Greece) [62], and PC12-htau cells stably transfected239
with the human tau (htau; 3 R/0 N isoform) trans-240
gene and expressing hyperphosphorylated tau (a kind241
gift of Dr I. Sotiropoulos, Life and Health Sciences242
Research Institute (ICVS), School of Health Sci-
243
ences, University of Minho, Portugal)[63], were used244
as in vitro models of AD. All cells were cultured
245
in Dulbecco’s Modified Eagle’s Medium (DMEM)
246
without L-glutamine, maintained at 37C in a humid-247
ified 5% CO2environment. For SH-SY5Y cells,248
the medium was supplemented with 10% (vol/vol)249
heat-inactivated fetal bovine serum (FBS), 1% antibi-250
otic/antimycotic (10,000 units/mL of penicillin,251
10,000 g/mL of streptomycin, 25 g/mL of ampho-
252
tericin B) and 1% L-alanyl-L-glutamine. For SH-253
SY5Y-APP cells, the SH-SY5Y culture medium
254
contained an additional 100 g/mL of G418 (Gibco,255
Thermo Fisher Scientific Inc.). For PC12 cells, the
256
DMEM without L-glutamine was supplemented with257
5% (v/v) heat-inactivated horse serum (HS) and the258
cells were plated on collagen-treated flasks/plates259
[64]. For PC12-htau cells the culture medium con-260
tained an additional 100 g/ml of G418 (Gibco,261
Thermo Fisher Scientific Inc.). Differentiation of SH-262
SY5Y and SH-SY5Y-APP cells was achieved with
263
the addition of all-trans retinal (Sigma-Aldrich Co.)
264
to the culture media, to a final concentration of 105
265
M for 6 days. For the differentiation of PC12 and
266
PC12-htau cells, 0.75% FBS, 0.75% HS, 100 ng/ml 267
7 S nerve growth factor (NGF; Invitrogen), 1% antibi- 268
otic/antimycotic and 1% L-alanyl-L-glutamine were 269
added to the media for 7 days. 270
Natural products cell viability assays 271
Differentiated SH-SY5Y or PC12 cells were 272
exposed to a range of concentrations of either extract 273
(ranging from 0.04 g/ml to 400 g/ml for each natu- 274
ral extract) for 24 h or 72 h. The effect of the exposure 275
to each extract/concentration on cell viability was 276
evaluated with the Water Soluble Tetrazolium Salt 277
-1 assay (WST-1; Takara), a colorimetric test based 278
on the cleavage of WST-1 by mitochondrial dehy- 279
drogenase and the measurement of the absorbance 280
of the resulting formazan product at 450 nm in an 281
ELISA reader (Lucy 2; Anthos Labtec Instruments 282
GmbH). Since both extracts were diluted in DMSO, 283
matched concentrations of DMSO were used as con- 284
trol for each product concentration. All experiments 285
were performed at least three times. 286
Immunoblotting 287
Cells were lysed in lysis buffer (50 mM Tris, 288
pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton) 289
supplemented with a mixture of protease inhibitors 290
(P8340; Sigma-Aldrich Co.), incubated on ice for 291
30 min and centrifuged at 13,000 rpm for 5 min. 292
For sAPPthe culture medium was collected and 293
condensed with centrifugal filter units (Amicon; 294
Millipore) at 4,000 rpm. The protein concentra- 295
tion was determined with the Bradford method 296
[65], using bovine serum albumin to generate a 297
standard curve. All samples were analyzed by SDS- 298
PAGE (Supplementary Table 1). GAPDH and actin 299
were used as loading controls in all cases, except 300
for sAPPwhere the volume of the initial cul- 301
ture media was used in sample normalization and 302
reciprocal sample volume loading. Proteins were 303
transferred to nitrocellulose membranes (Macherey- 304
Nagel GmbH & Co), which were then incubated with 305
primary antibodies (Supplementary Table 1). The 306
nitrocellulose membranes were subsequently washed 307
in 50 mM Tris-HCl, pH 7.5, 150mM NaCl, and 308
0.05% Tween 20 and incubated with a peroxidase- 309
conjugated anti-mouse (1:16,000 dilution; Sigma- 310
Aldrich Co.) or anti-rabbit (1:10,000 dilution; 311
BIO-RAD) secondary antibody. Protein signals were 312
detected using electrogenerated chemiluminescence 313
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(ECL) reagents according to the manufacturer’s pro-314
tocol (Thermo Fisher Scientific Inc.). The intensities
315
of the bands of interest from at least three dif-316
ferent experiments were quantified using Image J317
software (version 1.47v; https://imagej.nih.gov/ij/).318
The measurements for all phosphorylated forms were
319
normalized to the total levels of the corresponding320
proteins.321
Statistical analysis322
Descriptive statistics were performed for all
323
experiments and data obtained are presented as324
mean ±standard deviation (SD). A pvalue 0.05325
was considered statistically significant. For the
326
immunoblotting assays, the integrated densities of
327
the bands obtained were analyzed by Student’s t-test.328
The ratios of protein isoforms to total protein were
329
assessed by chi-square.330
RESULTS331
Plant extracts’ UHPLC-HRMS-ESI(-) profiles332
HRMS profiles were acquired using ESI, in
333
negative mode. Characterization of the detected
334
metabolites was achieved considering m/z val-
335
ues of the suggested elemental composition with336
a 5 ppm tolerance from the proposed theoretical337
mass, as well as RDB equivalent values (Fig. 1).338
A total of 40 secondary metabolites were iden-339
tified in the methanolic extract of S. scardica340
(Supplementary Table 2) and more than 30 com-341
pounds in the C. spinosum decoction (Supplementary
342
Table 3). The results of the analysis demonstrate
343
that the S. scardica extract is rich in phenylethanoic
344
glycosides, as well as flavonoids and their glyco-
345
sylated forms. The major components that were
346
detected under the class of phenylethanoic glyco-347
sides, were verbascoside, martynoside, echinacoside,348
lavandulofolioside, allysonoside, leucosceptoside,
349
forsythoside, samioside, as well as their iso-350
mers. Under the class of flavonoids, the main351
metabolites detected included scutellarein, isoscutel-
352
larein, hypolaetin, and apigenin, which were mainly
353
detected in their glycosylated forms, with or with-
354
out coumaroyl groups. The decoction of C. spinosum
355
appears to be rich in secondary metabolites belonging
356
to different chemical classes, including organic acids,357
such as citric, malic and cinnamic acid, flavonoid358
derivatives and sesquiterpene lactones. Caftaric, 359
cichoric and chlorogenic acid are the predominant 360
hydroxycinnamic acids in the extract, whereas in 361
the class of flavonoids, the principal compounds 362
were quercetin and luteolin glucuronides. Interest- 363
ingly, the extract appears to contain a small number 364
of sesquiterpene lactones such as the sulphonated 365
guianolide, 8-deacetylmatricarin-8-O-sulfate. 366
S. scardica and C. spinosum extracts do not 367
compromise viability of SH-SY5Y and PC12 cells 368
To evaluate the biological tolerance of neuron- 369
like cells for S. scardica and C. spinosum extracts, 370
we exposed them to a range of biologically rele- 371
vant concentrations for different time periods and 372
measured their effects on cell viability with WST- 373
1 assays [66–68]. In specific, differentiated wild type 374
SH-SY5Y and PC12 cells were exposed to con- 375
centrations ranging from 0.04 g/ml to 400 g/ml 376
for each natural extract (Figs. 2 and 3). Both S. 377
scardica and C. spinosum preserved intact the viabil- 378
ity of differentiated SH-SY5Y cells across all tested 379
concentrations and incubation times. These results 380
were confirmed by experiments in differentiated 381
PC12 cells. 382
Having determined that the specific S. scardica and 383
C. spinosum extracts do not compromise cell via- 384
bility, we proceeded to investigate their downstream 385
effects on APP processing and tau expression / phos- 386
phorylation in two AD neuronal cell culture models 387
(differentiated SH-SY5Y-APP and PC12-htau). 388
Effects on AβPP processing 389
Since APP processing is considered a central 390
pathogenetic mechanism for AD, we proceeded to 391
assess the effect of S. scardica and C. spinosum 392
on the expression of its key molecular players. Dif- 393
ferentiated SH-SY5Y-APP cells were treated with 394
the maximal non-toxic concentration, as determined 395
above, for 72 h (400 g/ml) and compared against 396
DMSO treatment. APP-C83 and sAPPwere used 397
as markers of the non-amyloidogenic alpha amyloid 398
pathway, while APP-C99 and -secretase (BACE1) 399
as markers of the amyloidogenic Apathway. The 400
levels of -secretases (PSEN1 and PSEN2), which 401
are implicated in both the alpha and beta amyloid 402
pathways, were also assessed (Fig. 4). 403
The C. spinosum extract significantly increased 404
APP-C99 by 73.3% and sAPPby 65.6%, thus 405
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Fig. 1. UHPLC-HRMS-ESI(-) profiles of: (A) S. scardica methanolic extract, and (B) C. spinosum decoction.
significantly altering the ratio of processed APP
406
peptides to total APP. Furthermore, it increased407
PSEN1-CTF by 69.7% and PSEN2-CTF by 166.4%,408
while it reduced BACE1 by 50.4% and PSEN1409
by 34.36%, overall affecting the ratio of processed410
PSEN1 and PSEN2 compared to their respective total411
protein levels.
412
The S. scardica extract significantly increased 413
cellular APP by 68.7%, PSEN1 by 146.7%, 414
PSEN1-CTF by 126.3% and PSEN2-CTF by 92.7%, 415
while decreasing BACE1 by 57.0% and the PSEN2 416
complexes by 36.1%. Overall it significantly affected 417
the ratio of processed APP peptides to total APP, 418
as well as those of PSEN1 and PSEN2. 419
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Fig. 2. Diagrammatic presentation of viability of differentiated SH-SY5Y cells at 24 and 72 h of treatment with different concentrations of:
(A) S. scardica extract, (B) C. spinosum extract (n=3, the concentration axis is log scaled).
Fig. 3. Diagrammatic presentation of viability of differentiated PC12 cells 24 and 72 h of treatment with different concentrations of: (A) S.
scardica extract, (B) C. spinosum extract (n=3, the concentration axis is log scaled).
Effects on tau phosphorylation420
Tau hyperphosphorylation is believed to be cru-
421
cial in AD pathogenesis by promoting the formation422
of NFTs and ultimately neural loss. Firstly, we con-423
firmed that tau is hyperphosphorylated at Thr231
424
and Ser199/Ser202 in PC12-htau cells compared425
to PC12 wt cells (Fig. 5). We then proceeded to
426
assess the effects of S. scardica and C. spinosum427
using differentiated PC12-htau cells expressing428
hyperphosphorylated human tau. The endpoints mea-
429
sured included tau phosphorylation (pThr231-tau and
430
pSer199/Ser202-tau), as well as expression and acti-
431
vation of the tau kinases GSK3and ERK1/2. It
432
should be noted that both GSK3and ERK1/2 were 433
activated in control PC12-htau cells, in agreement 434
with previous reports on AD (Fig. 6) [22, 69, 70]. 435
We found that treatment of differentiated PC12- 436
htau cells with the C. spinosum extract decreased total 437
tau (by 42%), the phosphorylation of tau (pThr231 438
by 77% and pSer199/Ser202 by 35%), ERK1 (by 439
38%), ERK2 (by 78%), pERK1 (by 55%), and 440
pERK2 (by 79%), while it increased the inactive 441
pSer9-GSK3(by 90%) compared to the DMSO 442
treatment. The S. scardica extract reduced total tau 443
(by 45%), the phosphorylation of tau (pThr231 by 444
75% and pSer199/Ser202 by 66%), ERK2 (by 35%) 445
and pERK2 (by 85%), and it increased the levels 446
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Fig. 4. Immunoblotting assessment of APP processing components after treatment of differentiated SH-SY5Y-APP cells with S. scardica
or C. spinosum extracts. A) Immunoblotting detection of cellular APP, APP-C99, APP-C83, sAPP, BACE1, PSEN1 complexes,
PSEN1, PSEN1-CTF, PSEN2 complexes, PSEN2, PSEN2-CTF, with GAPDH as internal control of protein expression. B) Diagrammatic
presentation of the percent change in expression of protein levels following treatment relative to DMSO control. Total APP protein levels
were based on the sum of the immunoblotting measurements of cellular APP, APP-C99, APP-C83, and sAPP. C) Diagrammatic
presentation of PSEN1 (top panel) and PSEN2 (bottom panel) processed forms to total protein levels. The size of the pie chart represents
the ratio of protein expression to total protein levels compared to DMSO control treatment of cells (*p< 0.05, t-test, n= 3).
Fig. 5. Immunoblotting assessment of tau hyperphosphorylation in PC12-htau cells. A) Immunoblotting detection of tau hyperphosphory-
lation at Thr231 and Ser199/Ser202 in PC12 and PC12-htau cells, with actin as internal control for protein expression. B) Diagrammatic
presentation of tau phosphorylation in PC12 and PC12-htau cells (*p< 0.05, t-test, n= 3).
Uncorrected Author Proof
I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease 9
Fig. 6. Immunoblotting assessment of the tau phosphorylation pathway components after treatment of differentiated PC12-htau cells with
S. scardica or C. spinosum extracts. A) Immunoblotting detection of pThr231-tau, pSer199/Ser202-tau, total tau, pSer9-GSK3, GSK3,
pERK1/2, total ERK1/2, with actin as internal control of protein expression. B) Diagrammatic presentation of quantified protein expression
(*p< 0.05, t-test, n= 3).
of inactive pSer9-GSK3(by 150%) and ERK1 (by447
24%). Overall, C. spinosum and S. scardica have sim-
448
ilar effects on all investigated components of the tau449
pathway, with the exception of ERK1 (Fig. 6).
450
DISCUSSION451
AD is characterized by the aggregation of amy-
452
loid plaques and the formation of NFTs. In search of
453
new therapeutic approaches against AD we screened
454
Sideritis scardica and Cichorium spinosum extracts,
455
as they are integral part of the Greek Mediterranean456
diet, rich sources of polyphenols (such as flavonoids457
and phenolic acids) and start to emerge as protective458
agents of memory and cognition [58, 59]. In order to459
determine the molecular effects of S. scardica and C.
460
spinosum we used two cell culture models of AD 461
and focused on key players of the APP and the 462
tau processing pathways, as they constitute promis- 463
ing targets against AD neurodegeneration and disease 464
progression [71–73]. 465
The first step in the evaluation of their therapeutic 466
potential requires the examination of their effects on 467
wild type cell viability. Towards this end, we used an 468
assay based on colorimetric tetrazolium salts cleav- 469
age to formazan by enzymes of metabolically active 470
cells [74]. Such assays are recommended because 471
they combine measurement of lethality, proliferation 472
and metabolism, and they are valuable for dose selec- 473
tion [75]. The assays were performed independently 474
for two types of differentiated neuron-like cells, origi- 475
nating from the well-established SH-SY5Y and PC12 476
cell lines. Both C. spinosum and S. scardica extracts 477
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10 I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease
were very well tolerated at the cellular level, main-478
taining cell viability unaltered. The highest tested
479
biologically relevant and viability favorable doses480
(400 g/ml) were used for in depth studies at the481
APP and the tau processing pathway levels.482
Differentiated neuron-like SH-SY5Y-APP over-
483
expressing APP695, were used to dissect the effects484
of the C. spinosum extract on the key steps of485
the amyloidogenic and non-amyloidogenic APP486
processing pathways. Notably, we observed signif-
487
icantly increased levels of sAPP, the product488
of the non-amyloidogenic APP pathway. It has489
been shown that sAPPhas neurotrophic, neuro-
490
protective, and synaptotrophic properties, enhances491
neurite outgrowth, LTP, and memory retention and
492
is involved in the proliferation of neural precursor493
cells [76–82]. In addition, sAPPhas been found to494
act as a neuronal metallotransporter and metallochap-
495
erone, regulating metal homeostasis, an important496
process for Apeptides cleavage [77]. The increase497
in sAPPis likely mediated by the observed signif-498
icant increase in the active components, PSEN1-CTF499
and PSEN2-CTF, of the respective gamma secretases500
[83]. Meantime, the levels of PSEN1 were signif-501
icantly decreased. This could be explained by its
502
increased cleavage to PSEN1-CTF, a decrease in
503
protein expression or a combination of both. Incu-504
bation with C. spinosum also led to the reduction of
505
BACE1 levels, the beta secretase mediating Apro-506
duction which is reported increased in the brain of AD507
patients [84]. Consistently with our findings, cichoric
508
acid, a compound detected in our C. spinosum extract,509
has been shown to prevent memory impairment and
510
amyloidogenesis, at least in part, through the reg-
511
ulation of BACE1 levels [54]. Despite the reduced512
levels of BACE1, we observed increase of APP-513
C99. Although the mechanism behind this is unclear,514
it could potentially be due to accumulation of APP-515
C99 because of its reduced processing to A,asa516
consequence of the enhanced involvement of gamma
517
secretases in the non-amyloidogenic pathway, as518
described above. Overall, the C. spinosum extract
519
appears to promote APP processing preferentially520
through the alpha, non-amyloidogenic pathway.521
We proceeded to evaluate the effects of C.522
spinosum on tau hyperphosphorylation, using523
differentiated neuron-like PC12-htau cells which524
over-express hyperphosphorylated tau. Notably, a525
significant decrease of 42% was observed in total
526
tau levels. Tau proteins not only accumulate in the
527
brains of AD patients, but they are also emerging
528
as an informative predictor of a person’s cognitive
529
decline and potential response to treatment [85]. 530
Furthermore, the observed significant decrease in 531
phosphorylated pT231-tau and pSer199/Ser202-tau 532
by 77% and 35%, respectively, suggests a pro- 533
tective effect against phopsho-tau mediated AD 534
pathogenesis. 535
Since the phosphorylation of tau is primarily reg- 536
ulated by glycogen synthase kinase 3-beta (GSK3)537
and ERK1/2, we proceeded to assess both of these 538
pathways [22, 73, 86]. Treatment of the neuron-like 539
PC12-htau cells with C. spinosum led to signif- 540
icant increase of the inactive pSer9-GSK3, and 541
significant decrease of ERK1/2, as well as its 542
phosphorylated active forms pERK1 and pERK2. 543
Activation of GSK-3has been associated with the 544
formation of NFTs and the production of A[69], 545
while inhibition of GSK3through phosphoryla- 546
tion on Serine 9 (pSer9-GSK3) is neuroprotective 547
[22, 69, 70]. Activation of ERK1/2 increases both 548
tau phosphorylation and abnormal tau deposition in 549
AD, whereas inhibition of the ERK1/2 pathway pre- 550
vents tau-mediated cell death [87–90]. Consistently 551
with our observations, quercetin-3-O-glucuronide, 552
a compound detected in our C. spinosum extract, 553
has been shown to suppress the phosphorylation of 554
ERK1/2 and significantly reduced the generation of 555
Apeptides by primary neuron cultures [57, 91]. 556
Consequently, the C. spinosum extract, appears to 557
have a favorable impact on multiple steps of the tau 558
phosphorylation pathway, suggesting an overall neu- 559
roprotective effect. 560
The S. scardica extract also presented with sig- 561
nificant effects on the APP processing and tau 562
pathways. In specific, treatment of differentiated SH- 563
SY5Y-APP cells led to a significant decrease in 564
BACE1. This is in agreement with the reported 565
down-regulation of BACE1 by apigenin, a compound 566
detected in our S. scardica extract, which has been 567
shown to suppress amyloidogenesis and to amelio- 568
rate AD-associated learning and memory impairment 569
[44, 92]. BACE1 has been shown to regulate the lev- 570
els of full length APP [93]. Consistently with these 571
findings, we observed a significant increase in cellu- 572
lar APP. Furthermore, the expression of the PSEN1 573
complexes, PSEN1-CTF and PSEN2-CTF were sig- 574
nificantly increased, whereas the PSEN2 complexes’ 575
levels were decreased. These findings, in combination 576
with in vivo studies showing a significant reduc- 577
tion in buffer soluble A42 and decreased amyloid 578
plaque formation (number and size) post S. scardica 579
treatment [45], support the down-regulation of the 580
amyloidogenic pathway. 581
Uncorrected Author Proof
I. Chalatsa et al. / Natural Products Against Alzheimer’s Disease 11
Importantly, S. scardica appears to also have a sig-582
nificant effect on tau related pathways, similar to this
583
of C. spinosum. In specific, treatment of PC12-htau584
cells resulted in a significant decrease of pThr231-585
tau, pSer199/Ser202-tau and pERK2, as well as the586
increase of the inactive pSer9-GSK3. These data
587
suggest that S. scardica could be a potential inhibitor588
of GSK3and ERK1/2 activation, and consequently589
of tau phosphorylation, all of which are directly590
implicated in AD pathogenesis. Indeed GSK3 is con-
591
sidered a promising therapeutic target for AD, and592
its inhibition by lithium reduced tau phosphoryla-593
tion in vivo and lowered the level of tau aggregates
594
[94, 95].595
Research on the potential of natural products in
596
preventing or treating specific diseases, including597
AD, is increasing exponentially [96]. Although there598
is substantial lack of scientific evidence or clinical
599
trials to support the use of natural products against600
AD, their market as nutritional supplements is boom-601
ing [96, 97]. Our findings demonstrate that the C.602
spinosum and S. scardica extracts cause significant603
molecular changes that could cumulatively reverse604
molecular processes associated with amyloidogene-605
sis and tau hyperphosphorylation. Furthermore, our
606
findings support and explain recent epidemiologi-
607
cal studies, suggesting the existence of a direct link608
between adherence to a Mediterranean style diet and
609
low risk for cognitive disease development [37–39].610
Recent data from the Hellenic Longitudinal Inves-611
tigation of Aging and Diet (HELIAD) suggest that
612
adherence to the Mediterranean diet has a neuropro-613
tective effect and is positively associated with better
614
cognitive performance and lower dementia rates in
615
Greek elders (prevalence of dementia in Greece is616
4.5%, the lowest in Europe) [98]. In conclusion,617
the Mediterranean style diet components Cichorium618
spinosum and Sideritis scardica extracts emerge as619
promising for the prevention and/or treatment of AD,620
through the inhibition of APP and tau misprocessing
621
pathways leading to AD.622
ACKNOWLEDGMENTS
623
We are grateful to Professor S. Efthymiopoulos,624
Professor L. Stefanis, Dr. K. Vekrelis, Dr M. Xylouri,625
Dr N. Koulakiotis, Dr I. Dafnis, and M. Chatzis-626
tavraki.627
This work has been supported by a “Large Scale628
Cooperative Project” (TreatAD, 09SYN-21-1003)
629
co-financed by the European Social Fund (ESF) and630
the General Secretariat for Research and Technology 631
in Greece. 632
Authors’ disclosures available online (https:// 633
www.j-alz.com/manuscript-disclosures/17-0862r1). 634
SUPPLEMENTARY MATERIAL 635
The supplementary material is available in the 636
electronic version of this article: http://dx.doi.org/ 637
10.3233/JAD-170862. 638
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